Single-cell Transcriptome Integration Analysis Of Inter-and Intra-tissue Heterogeneity In Human Intestinal Mast Cells

Background and objectiveMast cells（MCs）are important immune cells in intestinal tissues,and an increasing number of studies have identified MCs as the basis for the pathogenesis of several intestinal diseases,including inflammatory bowel disease（IBD）.Several recent single-cell RNA sequencing（scRNA-seq）studies have revealed an important role of MCs in inflammation,fibrosis and neoplasia.MCs have a very significant and complex heterogeneity influenced by multiple factors.Therefore,these studies of MCs in only a single tissue or disease type have not yet been able to reveal how MCs perform functions common to their lineage and required for their resident organs.The aim of this study was to create a single-cell atlas of human MCs by integrating and analyzing scRNA-seq data from multiple tissues and disease states.The focus was to explore the inter-and intra-tissue heterogeneity of intestinal MCs in healthy and disease states,and to provide a pathway and data resource for understanding the critical role of MCs in disease.MethodsA total of 29 publicly available scRNA-seq datasets were included in this study for data integration and comprehensive analysis using multiple bioinformatics techniques,including（1）data pre-processing,including Scrublet algorithm for preliminary doublets removal,quality control,normalization and logarithmization;（2）Harmony algorithm for data integration and batch effect correction;（3）principal component analysis and unified manifold approximationand projection algorithm for downscaling and visualization of data;（4）Leiden clustering analysis and cell type annotation;（5）differentially expressed genes and transcription factors analysis;and（6）GSEApy for pathway enrichment analysis.Meanwhile,key molecular targets were validated in human samples using immunofluorescence staining.Results1.This study established a single-cell atlas of human MCs across tissues and disease states containing 18,064 MCs.2.In healthy states,intestinal MCs shared a core transcriptional program capable of producing proteases,lipid mediators and histamine with MCs from barrier tissues such as lung,oral cavity and skin.Simultaneously,intestinal MCs exhibited tissue-specific characteristics:subsets of MCs with high expression of TMSB4X（P<0.001）and IL1RAPL1（P<0.001）genes were present in human colonic tissue samples.3.In healthy states,MCs were heterogeneous among different intestinal segments:duodenal MCs were highly expressed in IL1RL1（P<0.001）and TXNIP（P<0.001）,jejunal MCs were highly expressed in NACA（P<0.001）and FOSB（P<0.001）,ileal MCs were highly expressed in LMO4（P<0.001）,NR4A1（P<0.001）and BCL2A1（P<0.001）,colonic MCs were highly expressed in TMSB4X（P<0.001）and HSP70 family member genes.4.Compared to the healthy state,the expression of 20,9 and 121 genes were up-regulated in inflamed,uninflamed and fibrotic tissue MCs,respectively;two specific subgroups,Diseased-MC₅ and Diseased-MC₆,existed in MCs of barrier tissues in disease states,with the former highly expressing ISG15（P<0.001）and IFITM protein family genes and the latter highly expressing various proliferation-related genes such as MKI67（P<0.01）and TOP2A（P<0.001）.5.Compared with healthy tissues,60 and 31 genes were up-regulated in MCs in the inflamed and uninflamed regions of the colon in patients with ulcerative colitis,respectively.16 and 12 genes were up-regulated in MCs in the inflamed and uninflamed regions of the ileum in patients with Crohn’s disease,respectively.The expression levels of SLC18A2 and TMEM176B were significantly upregulated in MCs in the inflamed region of the colon in patients with ulcerative colitis（P<0.001;P<0.001）,and the presence of SLC18A2+MCs and TMEM176B+MCs was verified in human colon samples using immunofluorescence assays,and the density of TMEM176B+MCs was significantly increased in patients with ulcerative colitis（P<0.05）.Conclusion1.This study provided a resource of human MCs data across tissues and disease states,which was more beneficial for the exploration of MCs heterogeneity.2.In healthy states,compared with other tissues and organs,intestinal MCs exhibited significant tissue specificity and varied significantly among intestinal segments,which might be related to the unique functional needs of intestinal tissues and differences in the tissue microenvironment among intestinal segments.3.The disease state significantly altered the number and phenotype of MCs,which might be related to the influence and shaping of MCs by different disease microenvironments.4.The number and phenotype of MCs in inflamed areas of IBD patients were significantly altered,and these alterations were already partially present in MCs in uninflamed region of IBD patients,suggesting that MCs are involved in the process of IBD development and progression.