Single-Cell Transcriptome Reveals The Immune Landscape Of Lung Of HDM And LPS Induced Steroid-Resistant Asthma Exacerbation In Mice

Background and ObjectiveIn the twentieth century,asthma was associated with allergies and airway infiltration of eosinophils,leading allergic asthma the first and most characteristic asthma phenotype.Research at the time focused on therapy to reduce eosinophil infiltration and reduce accompanying Th2 inflammation.Therefore,steroid therapy is the best way to achieve this goal and become the cornerstone of asthma treatment.Although most asthma patients are currently able to effectively relieve symptoms after corticosteroid treatment,5-10%of asthma patients respond poorly to steroid treatment.These asthma patients often need to be hospitalized,placing a heavy financial burden on the healthcare system.Factors that cause exacerbation of asthma include house dust mites,pollen,respiratory infections,cigarette smoke and pollutants etc.Increasing evidence suggests that the interaction between the innate immune response and the potential type ? immune microenvironment in the lung changes the outcome of inflammation and triggers exacerbations.HDM-derived proteases promote the pathogenesis of asthma by promoting abnormal innate and adaptive immune responses.Lipopolysaccharide(LPS)is usually derived from cytoderm of Gram-negative bacteria and environmental contaminants.The level of LPS is directly related to the severity of asthma and decreased lung function.In addition,clinical studies and experimental models of allergic asthma and exacerbation of asthma have shown that LPS,along with allergens,may play an important role in exacerbating disease and transforming the glucocorticoid-responsive phenotype to a glucocorticoid non-responsive phenotype.Of note,neutrophil recruitment is one of the salient features of acute exacerbation in patients who respond poorly to glucocorticoid.Infiltration of neutrophils into the airway indicates that the host's innate immune defense mechanism is activated to fight infection.Although there is several evidence that respiratory infections and allergen exposure contribute to the development of asthma exacerbation,the intricate molecular network between neutrophil infiltration,glucocorticoid resistance,and the pathogenesis of airway obstruction during exacerbation of asthma is not yet clear.In recent years,asthma-based immunotherapy has greatly improved treatment strategies,but its efficacy has not been consistent among asthma patients or subtypes.In this regard,it is important to identify specific biomarkers and molecular networks that can not only predict treatment outcomes,but also unlock complex back and forth movements between host immune cells.Single-cell RNA deep sequencing(ScRNA Seq)has recently been applied to understand the development and interactions of heterogeneous cells and to detect the expression of a large number of genes and transcriptional subtypes across the genome.This enables us to study the highly complex pathological mechanisms of chronic diseases with unprecedented detail.Exaggerated airway hyperresponsiveness(AHR)and inflammation are features of asthma and usually reflect the extent of asthma exacerbation.LPS exposure is related to the severity of the disease and steroid resistance.To investigate the mechanism of asthma exacerbation,we established a mouse model of LPS-induced exacerbation in the context of house dust mite(HDM)-induced allergic airway inflammation.To reveal the complexity of asthma exacerbations,we used single-cell RNA deep sequencing to analyze immune cells in the lungs during exacerbations and examine the effects of dexamethasone treatment.MethodsMale BALB/c mice of 6-8 weeks were randomly grouped into normal saline control group,HDM+LPS group,HDM+LPS+ DEX group,HDM group,LPS group,HDM+ DEX group.HDM i.n.challenge to induce an AAD model.LPS i.n.challenge to aggravate asthma,and DEX was injected intraperitoneally.The Flexivent device was used to measure the airway resistance to methacholine to evaluate the lung function of the mice.After the lung function was measured,the bronchoalveolar lavage fluid(BALF)of the mice was collected and then we count the different types of cells in the BALF to evaluate the inflammation.The left lobe of lung was obtained for pathological tissue sections,was quantify the mucus-producing cells and eosinophils and the inflammation score by HE&PAS staining.Finally,we select the SAL group(normal saline control group),VEH group(HDM+LPS group),and DEX group(HDM+LPS+DEX group),use flow cytometry to sort out immune cells in mouse lung tissue for single-cell sequencing,and then perform bioinformatics analysis of the sequencing results.Results1 HDM and LPS exposure induces steroid-resistant asthma exacerbationCompared with the SAL group,the airway hyperresponsiveness in the VEH group was significantly increased(p<0.01);the number of eosinophils,neutrophils,lymphocytes and total cells in BALF was significantly increased(p<0.01),but the number of macrophages was not significantly different between the three groups;the number of mucus-producing cells and eosinophils and inflammation scores in the VEH group were significantly higher than those in the SAL group(p<0.01).Compared with the VEH group,the airway hyperresponsiveness level in the DEX group was comparable;the number of eosinophils,neutrophils,macrophages,lymphocytes and total cells in BALF did not decrease;there were no significant differences between the two groups in pathological sections.2 Combined exposure of HDM and LPS has more severe AHR and airway inflammation than exposure to HDM or LPS aloneCompared with the SAL group,the airway hyperresponsiveness in the HDM group increased(p<0.01);the number of eosinophils,neutrophils,lymphocytes and total cells in BALF increased significantly(p<0.01),and the number of macrophages was not significantly different between the four groups;the number of mucus-producing cells and eosinophils and inflammation scores in the HDM group were significantly higher than those in the SAL group(p<0.01).Compared with the HDM group,the airway hyperresponsiveness in the HDM+LPS group increased(p<0.01);the number of eosinophils,neutrophils,and lymphocytes in BALF increased significantly(p<0.01);the number of mucus-producing cells and inflammation score in the HDM+LPS group were significantly higher than those in the HDM group(p<0.01).3 Dexamethasone reduces AHR and airway inflammation caused by HDMCompared with the SAL group,the airway hyperresponsiveness of the HDM group increased(p<0.01);the number of eosinophils,neutrophils,lymphocytes and total cells in BALF increased significantly(p<0.01),and the number of macrophages was not significantly different between the three groups;the number of mucus-producing cells and eosinophils and the inflammation score in the HDM group were significantly higher than those in the SAL group(p<0.01).Compared with the HDM group,the airway hyperresponsiveness level of the HDM+DEX group decreased(p<0.01);the number of eosinophils,neutrophils,lymphocytes and total cells in BALF was significantly reduced(p<0.01);the number of mucus-producing cells and eosinophils and the inflammation score in the HDM+DEX group were significantly lower than those in the HDM group(p<0.01).4 ScRNA Seq analysis of lung immune cells identifies multiple immune cell clustersCD45+immune cells from mouse lung tissue mainly contain 20 cell clusters after single-cell sequencing,including multiple subsets of T cells,B cells,monocytes,macrophages,NK cells,dendritic cells,and neutrophils and basophils.The numbers of some types of cells were significantly different between the SAL group,the VEH group,and the DEX group.5 Neutrophils,monocytes,CD11b-macrophages,and NK cells may be associated with steroid-resistant severe asthmaWeighted gene co-expression network analysis(WGCNA)was used to assess the gene interactions between multiple cell clusters,and differential expressed transcripts were grouped in 6 co-expressed modules.Unsupervised hierarchical clustering analysis was used to cluster samples.Black modules was found in T cell clusters,blue modules was found in NK cell clusters,brown modules was found in B cell clusters,and red modules are mainly expressed in CD11b-macrophage clusters,and the extent of expression in CD11b-macrophages much lower,turquoise and yellow modules was found in multiple cell clusters,but have a stronger effect in monocyte clusters and neutrophil clusters,respectively.To assess the activation of these clusters of cells,we analyzed the activation scores of several groups of immune response and disease-related gene characteristics in a single cell.Neutrophils,CD11b+macrophages and monocyte clusters have higher scores for inflammation modules and steroid response modules;CD11b+macrophage clusters and some monocytes,neutrophils and NK cell clusters are related to the asthma module;neutrophil clusters,CD11b-macrophage clusters,and most monocyte clusters have higher severe asthma module scores.Two large cohort studies,SARP and U-BIOPRED,have identified 104 genes that are closely related to the pathogenesis of severe asthma.Among these genes,Olfm4 and Lcn2 were mainly detected in neutrophil cluster and their expression was steroid-resistant.Multiple genes associated with severe asthma were identified in neutrophils,monocytes,NK cells,and CD11b+macrophage clusters.These data suggest that the development of severe asthma is regulated by a variety of immune cells.6 IL-4 and IL-13 are differentially expressed by Basophils,ILC2 and CD8+memory T cellsIL-4 and IL-13 are key cytokines driving type 2 responses.Dexamethasone treatment could not inhibit the expression of IL-4 by CD8-DCs and basophils,and promoted the production of this cytokine by Ly6C+and Ly6C-monocytes,Tregs,and CD103-DCs.IL-13 was differentially expressed by CD8+memory T cells,ILC2 and basophils,which is not sensitive to glucocorticoids,and dexamethasone treatment slightly promoted the expression of IL-13 in CD8+memory T cells.7 Upstream regulators and Pathways analysis of Basophils,ILC2 and CD8+memory T cellsIn order to understand the activation of intracellular molecular networks of basophils,ILC2,and CD8+memory T cells during the exacerbation of asthma,we performed a gene set enrichment analysis using ingenuity pathway analysis(IPA).The subset of genes of the canonical pathways with highest activation score are mainly related to "EIF2 signaling",followed by "oxidative phosphorylation" and "Rho family GTPase signaling".Importantly,the activation of these pathways is glucocorticoid-resistant.Upstream regulator analysis predicted high activation of many upstream regulators,including STK11,NFE2L2,Hbb-b1,IFN?,CD38,and IL-5,in response to HDM/LPS stimulation.Except for TLR4 in CD8+memory T cells,TLR3/IL-1? in ILC2,and IL4/MYC in basophils,activation of most upstream regulators is steroid-resistant.ConclusionLPS can induce exacerbations of asthma caused by HDM and is resistant to glucocorticoids.We identified 20 major clusters of immune cell with different patterns by single-cell sequencing.A detailed examination of the characteristic genes of these cells revealed that expression of IL-4 and IL-13 by basophils,ILC2 and CD8+memory T cells is largely glucocorticoid resistant.This unprecedented detail resource is essential for studying the characteristics of the innate and adaptive immune cells in lungs and for developing effective treatment strategies for patients with exacerbation of asthma.