Study On Single-cell Transcriptomic Analysis Reveals Key Immune Cell Phenotypes In The Lungs Of Patients With Asthma Exacerbation

Background and objectiveAsthma exacerbations are usually characterized by increased inflammation,excessive airway mucus secretion,and airway hyperresponsiveness(AHR).If routine treatment is not effective,hospitalization is still needed.There are many common risk factors that increase susceptibility to asthma exacerbations,such as allergens,respiratory infections,smoking and air pollutants.The etiology of asthma exacerbations is heterogeneous due to the complicated network system of innate and adaptive host immune response.during acute exacerbation of asthma the multiple molecular networks that make up the abnormal immune response of the lungs remain unclear.Current treatment options are mainly glucocorticoids and β2 receptor agonists,which are effective for stable asthma and are poor in controlling symptoms in patients with acute exacerbation of asthma.It is an urgent problem to further clarify the pathogenesis of asthma in order to control the symptoms of this part of patients more effectively via targeting therapy or other new methods.More and more people recognize the importance of the network-based analysis method,which is an effective tool to analyze high-dimensional data of disease and extract huge amount of information on the gene association from biomedical research.Systematic network studies provide a fair way to understand the pathogenesis of asthma exacerbation.however,due to bulk tissue biopsy samples or limited predefined markers,previous multiomics studies have not been designed to identify molecular networks specific to individual immune cells in asthma exacerbation.which limits our ability to explain immune pathogenesis at the cellular level.In this study,we used BD Rhapsody platform-based high-throughput single-cell RNA-Seq,to capture large-scale immune cell mRNA in bronchoalveolar lavage fluid(BALF)in patients with asthma exacerbation.the pathophysiological mechanism of asthma exacerbation was explored by network analysis from single cell level.MethodsFrom February 2019 to June 2019,11 asthma patients and 5 healthy controls were recruited from the first affiliated Hospital of Zhengzhou University.Flow cytometry was used to sort out immune cells from alveolar lavage fluid,living goal cells are performed to single cell RNA sequencing,and then we analyze sequencing results by bioinformatics analysis.Results1.Clinical characteristics of subjectsOur study recruited 16 individual,including 11 asthma patients(male:n=5,female:n=6),5 healthy people(male:n=3,female:n=2).Average age of asthma patients was 42.9 years(standard error 4.08)and had impaired lung function,the mean of FEV1%predicted was 85.7(standard error 4.85),Average age of healthy controls was 44.4±5.99 years.Blood tests in the asthma group comparing to healthy controls was significant increasing in the total number of leukocytes(mean±standard error was 7.313±0.5688x 109/L),the mean value of neutrophil,eosinophil,monocyte and lymphocyte were higher than those of healthy controls respectively.Both serum IgE and exhaled nitric oxide levels were significantly higher than those in healthy controls.Six patients in the asthma group had more than two exacerbations in the past year,5 cases with 1 or 2 acute exacerbation;In the past year,5 patients were hospitalized for acute exacerbation of asthma;There were 2 cases of acute exacerbation of asthma in the past year.2.Immune cells in BALF were sorted out by flow cytometryBALF was processed into single-cell suspension,and CD45+7AAD-cells were sorted out by FACS ARIAIII.The cell viability of each sample was more than 90%after sorting,and the total number of cells in each sample was more than 1×105.The cell suspension of the object was observed under microscope without cell agglomeration,cell debris and impurities.3.Single cell RNA sequencingCD45+cells in BALF from 11 asthma patients and 5 healthy subjects were sequenced by using a single-cell sequencing system.Single cell samples were inspected and captured before library construction.Cell diameter distribution measured by Countess II was recorded,and then single cell sorting labeling was performed.The whole process was scanned by BD Rhapsody imaging system to evaluate the actual placement of cells and beads.Each step passed the quality control.4.Single-cell transcriptome sequencing of immune cells in the lung identifies multiple immune cell subpopulationsBy analyzing CD45+immune cells from human BALF,we used SingleR to identify 69 different cell clusters with specific molecular markers.Each cell is classified into one of the following cell types:B cells,monocytes derived dendritic cells,alveolar macrophages,monocytes derived macrophages,monocytes,neutrophils,NK cells,CD4+T cells,or CD8+T cells.Interestingly,we also detected a small number of bronchial epithelial cells,stem cells(HSC-CD34+)and progenitor cells(common myeloid progenitor cells and granulocyte-monocyte progenitor cells[GMP]).We identified The activation status of these cells.5.Asthma exacerbation results in different cell clusters in bronchoalveolar lavage fluidAfter analyzing the above cell clusters,we further classified the cells into 16 major cell clusters.Compared with healthy controls,levels of multiple cell clusters in BALF were significantly increased in patients with asthma exacerbation,including cluster 6(C6)(macrophages:monocyte source:IL-4/DEX/TGF-β)and C7(monocytes:Salmonella-Penicillium-flagella).Some macrophage clusters(C0)and CD8+T cells(C1)were also elevated.To detect the expression of proinflammatory cytokines,we examined the cytokine profiles of the identified cell clusters.Except for CCL5,which is expressed in both C1 clusters(CD8+T cells)in healthy controls and patients with asthma exacerbation,the cell clusters in healthy subjects did not produce significant changes in proinflammatory cytokine levels.In asthma patients,different cell clusters differentially expressed different cytokines.6.Intracellular signaling activity analysisIntracellular transcriptional regulation is an important means to regulate the proinflammatory response of immune cells.Analysis of 32 important transcriptional regulators showed that these factors were differentially expressed in 16 cell clusters in patients with asthma exacerbation comparing with healthy controls.C1(CD8+T cell cluster)strongly expressed IKZF3,RELA,RNUX3,STAT4,STAT5A/5B,and TBX21,while other transcriptional regulators were relatively weakly expressed.C3(macrophages:monocyte source:M-CSF/IFN-γ)strongly expressed SPI1 and STAT1,while healthy control group had low expression of SPI1 and STAT1.C15(neutrophils)highly expressed BCL6,RUNX1,and RUNX3,while C12(memory B cells),C13(NK cells),and C14(monocytes:CD16’)strongly differently express most regulatory factors.In contrast,C2(T cells:CD4+effector memory cells),C5(CD4+central memory T cells),C7(monocytes:Salmonella-Penicillium-flagella),C10(leukin-D4),and C11(T cells:y δ)clusters were weakly expressed for most transcriptional regulators.Except for BCL6 expressed by C15(neutrophils),SPI1 and BCL6 expressed by C0(macrophages:alveolar),C3(macrophages:monocyte source:M-CSF/IFN-y)and C9(monocytes:anti-FcGriib),most of the transcriptional regulators were not expressed in healthy control group.These data suggest that a large number of intracellular signaling pathways are activated during acute exacerbation of asthma to modulate the role of different immune cells.7.Expression of SARP and U-BIOPRED genesSARP and U-BIOPRED,two large cohort studies of asthma,have identified 104 genes that are strongly associated with the development of severe asthma.We found that 10 genes were significantly differentially expressed in the identified cell clusters.Among these genes,CEACAM8 and DPYSL3 are mainly expressed by macrophage clusters.AMPD3,ATXN7L1 and CHML were differentially expressed in macrophages,CD4+T cells and CD8+T cells.GSDMB was strongly expressed in T cell clusters,and Homer2 was expressed in monocytes.IL18R1 was mainly found in CD8+T cell clusters.What’s more,we were able to detect a partial expression of TSLP in the macrophage cluster.8.Genetic network analysisWe found significantly elevated levels of monocytes,CD8+T cells,and macrophages in the alveolar lavage fluid of bronchial asthma.In order to understand asthma exacerbations during the activation of intracellular molecular network,we choose C1-a(CD8+T cells),C6(macrophages:mononuclear cells source IL-4/Dex/TGF beta),C7(monocytes:salmonella-penicillium-flagella),CX-1(a macrophage subgroup:alveolar)and CX-2(a macrophage subgroup:alveolar),use "original pathway analysis"(IPA)"classical pathway","upstream regulatory factor" "disease and function" option on the gene enrichment analysis.Among them,C1-a(CD8+T cells)and C7(monocytes:Salmonella-Penicillium-flagella)had more upstream regulators and more active functions.The subset of genes in the classical pathway with the highest activation Z-score in C1-A and C7 is mainly associated with the"eIF2 signaling",followed by the ephrin receptor pathway,the "Rho family GTPase signaling pathway",and the CXCR4 pathway.All of these pathways are associated with infection.Conclusions1.The levels of macrophages,monocytes and CD8+T cells in the alveolar lavage fluid of patients with asthma exacerbation were significantly increased.2.Most of the proinflammatory cytokines in the alveolar lavage fluid of healthy subjects did not change significantly,while different immune cell clusters expressed different cytokines in the alveolar lavage fluid of patients with asthma exacerbation.3.During asthma exacerbation,a large number of intracellular signaling pathways are activated to regulate the functions of different immune cells.4.Monocytes and CD8+T cells were more active in alveolar lavage fluid of patients with asthma exacerbation.The gene subset of the classical pathway with the highest activation Z-score was mainly related to "eIF2 signaling",followed by the ephrin receptor pathway,the "Rho family GTPase signaling pathway" and the CXCR4 pathway.