Relationship Between TLR7 Positive Epidermal Cells And The Recurrence Of Keloids And The Mechanism

Background:Keloid is a locally aggressive benign fibrotic skin disease^[1].The pathogenesis has not been clarified yet.The mainstream therapy for keloid is the combination of the surgical resection and adjuvant radio therapy^[2,3].As an important part of lesion,the epidermis of keloid is involved in many ways to promote the formation of keloid.Epidermal pathological changes are funded in genes,protein expression,function and general morphology.The histological alteration of the epidermis includes increasing thickness,the flatting of the epidermal ridge,abnormal differentiation,and the twisted expression of surface markers^[4].Keloid-derived keratinocytes promote keloid-derived fibroblast,even normal skin fibroblasts,cell growth and migration by paracrine^[5-7].Sequential microarray analysis reveales the up-regulation of genes related to wound repair and mesenchymal changes in the epidermis of keloid,indicating that the epidermis is more directly involved in the formation^[8].Methods:This project intends to explore the possible role of TLR7-positive epidermal cells in the pathogenesis of keloids by exploring the phenotypic characteristics and cell functions of keloid epidermis.The main methods include multicolor flow cytometry analysis and a computer unsupervised automatic analysis pipeline,and cell proliferation experiments,migration experiments,quantitative detection of cell secreted proteins and in vitro scar models,observation of effect of TLR7 on keratinocyte cell behavior to understand how TLR7 participates in the pathogenesis of keloids from the cell phenotype.Results:1.We set up an optimized 9-color flow cytometric panel and a pipeline of SPADE on vi SNE to analyze the database.This pipeline was able to cluster the differences between keloid and normal skin epidermis.2.The abundance of cluster05(CD49f^hi/CD29^mid/TLR7^neg/CD24^neg),18(CD49f ^mid/CD29^mid/TLR7^neg/CD24^pos),23(CD49f^hi/CD29^hi/TLR7^neg/CD117^hi)decreased in the ear,compared with the abdomen,10.62,13.19,and 24.23-fold.The abundance of cluster15(CD49f^mid/CD29^mid/TLR7^mid)and 24(CD49f^hi/ CD29^hi/TLR7^hi)increased in the ear.3.The percentage of CD49f^mid-hi/CD29⁺/TLR7⁺/CD24⁺increased in the keloid epidermis,while the percentage of CD49f^lo-mid/CD29^-/TLR7^-/CD24^-decreased.4.The expression of CD49f⁺/CD29⁺/TLR7⁺subgroup increased in the keloid epidermis.CD49f expressed in the basal layer and upper basal layer of the normal epidermis.The fluorescence intensity was higher near the basal membrane layer.The expression pattern of CD29 was similar to CD49f,which was continuously expressed in the basal layer.TLR7 was continuously expressed in the basal layer but the expression intensity was uneven.5.IMQ activation of TLR7 promoted the migration of keratinocytes and keratinocyte epithelial anaplasia but had no obvious effect on the proliferation ability.TLR7-positive keratinocytes promoted fibroblast proliferation,collagen secretion and cytokine secretion.Conclusion:In this study,a nine-color flow cytometric panel was established to analyze the phenotypic changes of keloids.Through the observation of keratinocytes and fibroblast proliferation,migration,cell secretion ability,it shows that TLR7 has a promoting effect on the proliferation ability of keratinocytes and the change of epithelial mesenchyme transition.TLR7 positive keratinocytes contribute to the formation of fibroblast collagen,the secretion of cytokines and cell migration.It is suggested that TLR7 participates in the formation of keloids through various ways,and TLR7 may become an auxiliary diagnostic marker for the diagnosis of keloids and a new therapeutic target.