Research Of The Cellular Heterogeneity Of Keloid Based On Single Cell RNA Sequencing

Background:Keloid(KL)is a disease caused by fibroblast proliferation and excessive collagen accumulation,the incidence of which is still unknown and severely affects the quality of life of patients due to its painful and pruritic symptoms.Current treatment options for KL are limited and ineffective,and it is critical to investigate the pathogenesis to find more effective treatments.Single-cell transcriptome sequencing is a gene expression profiling assay at the single-cell level,which rapidly and precisely clarifies the gene expression patterns of thousands of cells.In this study,we used single-cell RNA sequencing technology and bioinformatics analysis to explore the cellular compartmentalization,cellular heterogeneity and potential functions of KL tissues to provide more insight into their physiopathological mechanisms and identify potential therapeutic targets.Objective:In this study,we use single-cell sequencing technology and bioinformatics to analyze the skin tissues of KL patients and normal human controls,with the aim of finding cell subpopulations and related genes associated with KL pathogenesis and exploring the pathogenesis and potential therapeutic targets of KL.Methods:In this study,single-cell RNA sequencing was performed by the 10 X Genomics Chromium TM system on the tissues of 3 KL patients and 2normal skin tissues,and the sequencing data were analyzed by bioinformatics to reveal the tissue and cellular heterogeneity of KL,including the roles of fibroblasts,vascular endothelial cells and immune cells in KL.(1)Collected lesional skin tissues from 3 KL patients and 2 normal human controls and single-cell RNA sequencing was performed.(2)Through bioinformatics analysis,the unsupervised clustering method and Seurat package were used to cluster and downscale the single cell data and annotate the cell subgroups;Monocle 2 was used to conduct the Pseudotime;the differentially expressed genes of different cell subgroups were screened and functional analysis of the differential genes was performed;cell communication analysis was used to understand cell interactions.(3)Verification of the pro-inflammatory fibroblasts and vascular endothelial cells in KL and normal human tissue sections by immunofluorescence staining.Results:1.Cell type identification.(1)The 21,953 cells from keloid tissues and 21,505 cells from normal human tissues were analyzed.According to single cell sequence,9different cell subgroups were identified in HC and KL,including Lymphocyte(Lym),Antigen presenting cell(APC),Mast,Keratinocyte(KC),Fibroblast(FIB),Smooth muscle(SMC),Vascular endothelial cell(VEC),Lymphatic endothelial cell(LEC)and neurocyte(NC)subpopulations.Fibroblasts were the most represented cell subpopulation in both HC and KL.(2)Vascular endothelial cell subgroups: Vascular endothelial cells(VECs)were identified,including VEC1,VEC2 and VEC3 groups based on known markers.(3)Fibroblast subgroups: Fibroblasts were identified 4 different subpopulations,including secretory-papillary fibroblasts(sp F),secretory-reticular fibroblasts(sr F),pro-inflammatory fibroblasts(pi F),and mesenchymal fibroblasts(m F)based on the known marker genes of each cell subgroup.(4)Immune cell subgroups: Lymphocytes and antigen-presenting cells are subgrouped according to the known marker genes of each cell subgroup.a.Lymphocytes were identified 7 different subpopulations,including including T helper cells(CD4+ T cells,Th),Granulozyme B-T cells(Gzmb-T),Plasmocyte(PC),Natural kill cell(NK cell),Cytotoxic T cells(CTL),Innate lymphocytes(ILC),and regulatory T cells(Treg).Among them,Th accounted for the largest proportion(58.1% in HC and 57.5% in KL),and there were more Treg,CTL,PC,and NK in KL than in HC,and less Gzmb-T than in HC.b.Antigen-presenting cells can be divided into: Dendritic cell 1(DC1),Dendritic cell 2(DC2),Migratory DC,and Macrophage(Macro)subpopulations.Among them,macrophages accounted for the largest proportion and there were more macrophages in KL than HC(52.9% of KL macrophages and 37.7% of HC macrophages),while there were fewer DC2 and migratory DCs within KL than HC and roughly equal proportions of DC1.2.Differential gene screening.(1)Total population differential expression genes: The differences of DEGs numbers in vascular endothelial cells were greatest in KL and HC,followed by fibroblasts and antigen-presenting cells.Genes significantly expressed in KL,such as VEGF and TGF,showed upregulation,while MHC class II molecules such as HLA-DQ,HLA-DP,and HLA-DR,as well as chemokines such as CXC and CC,showed downregulation.(2)Vascular endothelial cell differential expression genes: Compared to HC,CD39(ENTPD1),CD73(NT5E),and HIF1 A were significantly upregulated in vascular endothelial cells of KL.ICAM1,SELE,SOD2,GBP2,SERPINE were more highly expressed in VEC3 subpopulation.PECAM1,EMCN,PRCP,IL33,ACKR1,SNCG in VEC2 subpopulation,MCAM,HEY1 were more highly expressed in VEC1.(3)Fibroblast differential expression genes: The DEGs of fibroblast in KL were mainly concentrated in the pi F subpopulation,in which growth factors such as VEGF,TGF,HGF,collagen such as COL1A1,TNFRSF6 B,HGF,CD83 expression was upregulated,and chemokines such as CXC and interleukins were downregulated.And IL33 expression was mainly up-regulated in HC.(4)Immune cell differential genes.a.TGFB1 and TNFAIP3 were upregulated in Th and CTL subpopulations of lymphocytes,and various chemokines such as CCR7,CCL4,CXCR4 and CXCR6 were upregulated in Gzmb-T;ANXA1 and TNFSF14 were downregulated in Th subpopulation;S100A11 was downregulated in CTL.b.MHC class II molecules such as HLA-DQ are at low level in DC2,migratory DC and macrophage subpopulations of APC.Growth factors such as IGF and VEGF and pro-inflammatory factors such as IL10 were highly expressed in macrophages.Among the cell subpopulations,the cells with the highest number of DEGs were macrophages.3.Analysis of differential gene GO enrichment.(1)GO enrichment analysis of the total population: The upregulated genes were mainly enriched in extracellular structure organization,growth factor response,vascular development,angiogenesis and c positive regulation of cell motility and adhesion.The down-regulated differential genes were mainly enriched in p regulation of signal transduction by p53 class mediator,antigen presentation,regulation of T cell activation and intrinsic apoptotic signaling pathway.(2)GO enrichment analysis of vascular endothelial cells: The upregulated genes in endothelial cells were mainly enriched in Blood vessel development,blood vessel morphogenesis and angiogenesis.(3)GO enrichment analysis of fibroblasts: GO enrichment analysis of DEGs in KL fibroblast subpopulations revealed that the upregulated DEGs mainly enriched in the pi F subpopulation,as well as in the extracellular structure organization,collagen biosynthesis and modifying enzymes,blood vessel development.(4)GO enrichment analysis of immune cells: GO enrichment analysis of immune cell DEGs showed that the upregulated DEGs mainly enriched in Adaptive immune system,positive regulation of cell adhesion,negative regulation of immune system process,while down-regulated DEGs mainly enriched in regulation of T cell activation,immune effector process,cell activation and other pathways.There are a large number of differential genes enriched in cellular responses to stimuli,such as Gzmb-T,CTL,ICL,DC1,DC2,Migratory DC,and macrophages.4.Pseudotime analysis: Analysis of the differentiation trajectory of fibroblasts by the Monocle 2 revealed that fibroblasts in KL had two main differentiation directions,with state2 dominated by pi F and state3 dominated by m F.Compared with KL,fibroblasts in HC were mainly concentrated in state1,the primitive undifferentiated state.5.Immunofluorescence validation: It was confirmed by immunofluorescence technique that keloid tissues have a higher number of endothelial cells and more fibroblasts pi F subpopulation compared to normal skin tissues.Conclusion:1.KL cell profiles were mapped using single-cell RNA sequencing,and difference gene expression between different cell subpopulations in KL and HC were identified.2.fibroblasts and vascular endothelial cells accounted for a higher proportion of cells in each KL population and a higher number of DEGs,so we hypothesized that fibroblasts and vascular endothelial cells may be intimately involved in the pathogenesis of KL.3.The fibroblasts in KL differentiate mainly to the pi F subpopulation and m F subpopulation,and the pi F subpopulation in fibroblasts plays a dominant role in KL pathogenesis by promoting extracellular matrix synthesis through upregulation of growth factors such as VEGF and TGF and collagen such as COL1A1;participating in the formation of an immunosuppressive state in KL through upregulation of TNFRSF6 B,HGF,and CD83 expression;and promoting the formation of an immunosuppressive environment in KL through IL6 upregulation to promote the anti-inflammatory effect of macrophage M2 polarization.4.KL vascular endothelial cells promote the formation of an immunosuppressive state by upregulating the expression of HIF-1α,CD39(ENTPD1)and CD73(NT5E),and these genes can be used as targets for the development of KL disease.5.Th,CTL and macrophage subpopulations of KL immune cells were involved in the formation of an immunosuppressive state in KL by upregulating the expression of TNFAIP3,while promoting fibroblast proliferation.31 figures,9 tables,251 references.