IRF1-mediated Intermediate Cells Orchestrate Differentiation Of Epithelial Cells From Nasal Polyps

Objective:Aberrant differentiation of epithelial cells（ECs）promotes the occurrence and development of chronic rhinosinusitis with nasal polyps（CRSwNP）.However,the specific mechanism remains unclear.Objectives:To construct a detailed single-cell atlas of ECs from CRSwNP and explore the mechanism of aberrant ECs differentiation in the pathogenesis of nasal polyps.Methods:1.We generated sc RNA-seq profiles for nasal lavage fluid obtained from 3 CRSwNP patients and 3 control subjects.2.cells isolation from nasal lavage fluid were loaded onto a Chromium Controller to capture single cells and constructed sc RNA-seq library（10×Genomics）.3.The raw sc RNA-seq data were processed by demultiplexing,barcode processing,gene counting,and quality control.Then,a gene-barcode matrix was generated by principal component analysis and dimension reduction was performed to visualize the subset of datasets.4.Differential gene expression analysis was performed for clusters generated at various resolutions by both the Wilcoxon rank sum test and Model-based Analysis of Single-cell Transcriptomics.We then annotated the clusters by using the marker genes of the following major cell types.The ECs were re-clustered and re-annotated to identify subclusters.5.For investigation of potential interactions across different cell types among the epithelial cells,a cell-cell communication analysis was performed,which is a publicly available repository of curated receptors and ligands that can be used to detect ligand-receptor interactions.6.Trajectory analysis was performed for the ECs to determine the potential lineage between diverse EC phenotypes.Genes with branch-dependent expression dynamics were calculated by using the Branched Expression Analysis Modeling（BEAM）test in Monocle.7.Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis were performed for differentially expressed genes（DEGs）.We tested for significant DEGs in all target lists within a panel of annotated gene databases to achieve functional and pathway enrichment.8.Single-cell Regulatory Network Inference and Clustering（SCENIC）analysis was performed by using calculation of regulon activity scores with Area under curve（AUC）values and evaluation in each cell type to ascertain their activity.9.differentially expressed genes during ECs differentiation were identified with validation using proteomic analysis,immunofluorescence,and flow cytometry.Results:1.We profiled a total of 29 393 single cells detected in the 6 nasal lavage fluid samples of CRSwNP patients and control patients after quality control.2.Diverse epithelial and immune cell subtypes were identified,and 11 subtypes of ECs were re-identified.3.More than 140 differentially expressed genes were screened between the CRSwNP group and control group in each epithelia subgroup;among which,Cystatin SN（CST1）and Gelsolin（GSN）were the core up-regulated DEGs in all subgroups.4.An aberrant differentiation trajectory of ECs initially by pseudotime analysis appeared in a subpopulation of MUC5AC⁺FOXJ1⁺cells（intermediate cells）characterized by low expression of transcription factor Interferon Regulatory Factor 1（IRF1）in the CRSwNP group.5.a decrease of IRF1 also appeared in FOXJ1⁺PIFO⁺POS1^-ECRG4^-ciliated cells（ciliated2 cells）which were mainly enriched in aberrant differentiation trajectory immediately following intermediate cells.6.IRF1 dominated aberrant differentiation from intermediate cells to ciliated 2 cells by targeting downstream signaling molecules（IFI6,GBP1,PSMB8,PSMB9,and RARRES3）in CRSwNP.Conclusion:IRF1,as a core transcription factor,is not only involved in the aberrant differentiation of intermediate cells,but also plays a continuous regulatory role in subsequent aberrant ciliated cell differentiation.The differentiation process from IRF1^lowintermediate cells to IRF1^lowciliated 2 cells directly leads to the dysfunction of ECs from CRSwNP,which help to develop new strategies for treating CRSwNP.