Single Cell Transcriptomics Reveals Profiles Of Immune Cells In The Lungs Of Mice By HDM And CRA Inducing Asthma Exacerbations

Background and ObjectiveAsthma is one of the most common chronic inflammatory diseases of the lungs in my country.The main features are airway hyper responsiveness（AHR）,inflammatory cells infiltration in the lungs and increased mucus in the bronchial lumen.Asthma exacerbations present as acute progressive exacerbations of asthma symptoms and are a leading cause of hospitalisation and death in patients with asthma.Among adult asthma patients who have received anti-asthma medications,the incidence of asthma exacerbations is as high as 34%to 91%.The incidence of exacerbation of asthma is significantly related to allergen exposure.Common allergens include house dust mites（HDM）and cockroaches（CRA）.Exposure to the above two allergens can cause both innate immune response and adaptive immune response.The immune cells involved mainly include CD4⁺T cells,type 2 innate lymphoid cells,eosinophils and neutrophils,But the pathogenic mechanism has not been fully studied.Therefore,in this paper,we intend to use Single cellRNA Sequencing（ScRNA-Seq）to study the role of immune cells in the process of exacerbating asthma,and to further clarify the pathogenic mechanism of HDM and CRA induced asthma exacerbation.We looking forward to providing certain theoretical guidance for the treatment of clinical asthma exacerbations.Methods1.Choose 6-8 weeks old BALB/c male mice,and the mouse asthma exacerbation model was constructed by intranasal exposure to HDM and CRA allergens.The experiment was divided into four groups:PBS group（control group）,HDM group（asthma remission group）,HDMH group（HDM induced asthma exacerbation）,HDMC group（CRA induced asthma exacerbation）.2.This study intends to verify whether the asthma exacerbation model is successfully constructed from the following aspects:through bronchial provocation test in mice,the airway resistance of mice is measured,we determined whether the mouse had airway hyperresponsiveness;Bronchoalveolar lavage fluid（BALF）was collected immediately following lung function measurements,as previously described,through May-Grunwald-Giemsa staining（MGG）to assess the inflammation in the lungs of mice;Hematoxylin-Eosin staining（HE）was performed on the lung tissue sections of the left lobe of the mice,and score inflammation;q PCR technology was used to detect the relative expression of mRNA of mucin 5AC（Muc5ac）in mouse lung tissue.3.After the mice model was successfully constructed,the lung tissues of the three groups（PBS group,HDMH group,HDMC group）were prepared into single cell suspensions,the 7-AAD and CD45 antibody were added,the 7-AAD^-CD45⁺cells were sorted by using a flow sorter,and single cell transcriptome sequencing was performed.The data obtained from the sequencing was analyzed by bioinformatics.Results1.HDM and CRA induced asthma exacerbation in miceFrom the bronchial provocation test,the counts of inflammatory cells（eosinophils,lymphocytes,macrophages,neutrophils,etc.）in BALF,lung tissue pathology scores,and the relative expression of Muc5ac mRNA,the HDMC group and the HDMH group showed increased airway resistance,increased airway inflammatory cells,and increased Muc5ac gene expression,proving that the model was successfully constructed.2.ScRNA-Seq identifies multiple clusters of immune cell in the lungSince the HDM group was not restimulated with allergen within 15 days of constructing the asthma model,the level of asthmatic inflammation had decreased significantly and was not statistically different from the control group,therefore,we selected the control group and the two asthma exacerbation groups for subsequent single-cell sequencing analysis.Sort three groups of lung immune cells for single-cellRNA sequencing.The number of cells captured in each sample is greater than 10,000,and the median gene of each cell is greater than 1500.2)Perform mastering of highly expressed differential genes Component analysis（PCA）,screening 16 principal components for subsequent analysis.3)Use the Single R package to identify 20 main immune cell groups and 40 immune cell subpopulantions,including B cells,T cells,neutrophils,ILC2,NK cells,monocytes,etc..3.HDM and CRA induced asthma exacerbations lead to different immune cell populations in the lung tissues of miceThe counts of each immune cell were contrasted across groupscompared with the control group,both HDMC group and HDMH group had higher levels on Ly6C^-MHCII⁺monocytes,mature B cells,B-1a cells,CD4⁺T cells,and neutrophils;in addition to the above,the HDMC group had higher levels on DAP10^-NK cells and Ly6C^-MHCII^intmonocytes.However,in contrast to the other two groups,the HDMH group showed a significant decrease in the levels of T3 B cells,follicular B cells and a significant increase in the levels of neutrophils.4.Analysis of GO enrichment of genes in HDM and CRA induced asthma exacerbation in mice1)In terms of biological processes,compared with the PBS group,the genes of the HDMC group were significantly enriched in the positive regulation of cytokine production,the regulation of leukocyte adhesion,and the differentiation of T cells;While the genes of the HDMH group are significantly enriched in biological processes such as lymphocyte and T cell differentiation,T cell activation regulation,and the positive regulation of cytokine production.2)In terms of molecular function,the genes of the HDMC group and the HDMH group are significantly enriched in molecular functions such as ubiquitin,ubiquitin-like protein ligase,and GTPase binding.In addition to the above,the genes of the HDMC group are also significantly enriched in DNA Binding transcription factor binding function;and the genes of the HDMH group are also significantly enriched in phosphatase binding function.5.Analysis of KEGG enrichment of genes in HDM and CRA induced asthma exacerbation in miceWe performed KEGG pathway enrichment analysis on the selected differential genes,and showed the top 20 pathways where genes were significantly enriched.The results showed that compared with the control group,the genes in the HDMC group were significantly enriched in oxidative phosphorylation and NF-κB signaling pathways;while the genes of the HDMH group are significantly enriched in the three receptor signaling pathways of T cell,B cell,and C-type lectin,as well as the natural killer cell-mediated toxicity pathway.ConclusionHDM and CRA can successfully induce asthma exacerbations in mice,and we identified 20 major immune cell populations in mouse lungs by single-cell sequencing.Both HDM and CRA induced asthma exacerbations were associated with increased numbers of Ly6C^-MHCII⁺monocytes,mature B cells,B-1a cells,CD4⁺T cells and neutrophils;In addition to the above,CRA induced asthma exacerbations are associated with increased numbers of DAP10^-NK cells and Ly6C^-MHCII^intmonocytes;However,HDM induced asthma exacerbations were associated with increased numbers of neutrophils and decreased numbers of both T3 B cells and follicular B cells.