Study On The Pathological Mechanism Of Keloid Immune Microenvironment Based On Multi-omic

Background:Keloid is a skin fibroproliferative disease with unknown pathogenesis.The exploration of the mechanism of keloid and immune microenvironment based on multiple groups of technologies will be used as a new window for immunotherapy.Methods:We analyze the high-throughput data obtained by multiomics sequencing technology(metabolomics,transcriptome,single-cell sequencing)through RT-qPCR,Western blotting,immunohistochemistry and immunofluorescence,co-culture assay,flow cytometry,apoptosis assay,and animal model construction.The results of multiomics analysis were verified and the pathogenesis was explored.Results:Metabolomic analysis showed a tendency to decrease lipids and increase peptides in keloids compared with normal tissues.Important metabolites that differed significantly between the two groups included 1-methylniacinamide,4-hydroxyproline,5hydroxylysine,and L-prolyamide.Metabolomic analysis showed that 5-hydroxylysine and 1-methylniacinamide were metabolic indicators of keloid severity.The risk warning index of 5-hydroxylysine was 4×108-6.3×108(p=0.0008),and that of 1-methylniacinamide was 0.95×107-1.6×107(p=0.0022).Single-cell sequencing analysis showed that fiber cells,endothelial cells,mast cells,parietal cells and schwann cells increased obviously.The proportion of mesenchymal fibroblast subsets in keloid was significantly higher than that in surrounding normal skin tissue.In addition,we found significant differences in immune profiles between the two groups.The proportion of macrophages in keloids was significantly higher than that in surrounding normal tissue,while cDC2 cells were significantly reduced.Pseudotime trajectory analysis indicated two modules of macrophage cells(Module2:highly express RNASE1,C1QA,CD163,CD14,C1QC,FCGRT,MS4A7;Module10:highly express APOC1,CTSB,CTSL,TYROBP),which exhibited characteristics of tumor-associated macrophages(TAM),were upregulated in more advanced Keloids cells.Subsequently,analysis of cellular communication networks suggested that a macrophage-centered communication regulatory network may exist in keloids and that fibroblasts in keloids may facilitate the transition and proliferation of M2 macrophages,which contributes to further comprehension of the immunological features of keloids.The expression levels of costimulatory molecules(CD28,CD80,CD86 and CD40L)in the skin tissue of patients with keloids were higher than the levels in healthy people(p<0.05).The number and viability of fibroblasts cocultured with CD8+T cells were significantly reduced compared with those of the control(p<0.05).Patients with keloids who shows no signs of regression were found to have CD8+T cell immunosenescence.CD28,TNF,CD27 and IL2 associated with immunosenescent of CD8+T cell were decreased in keloid tissue compare to adjacent normal skin.The expression of CD28 and CD8+T cells as the input layer may be predictors of the severity of keloids with mVSS as the output layer.The high-risk early warning indicator for CD28 is 10-34,and the high-risk predictive indicator for CD8+T cells is 13-28.Conclusion:This study reveals the metabolic,transcriptomic and single cell features of keloid disease.Biomarkers of difference calculated by machine learning.Metabolomic analysis showed that 5-hydroxylysine and 1-methylniacinamide may be metabolic indicators of keloid severity.Abnormal expression of costimulatory molecules may lead to abnormal activation of CD8+T cells.Expression of CD28 and CD8+T cells as input layers may be predictors of keloid severity.In conclusion,in this study,we paint a picture of the immunology of keloid and provide new insights into its formation mechanism.