Research On The Mechanism Of Cartilage Degeneration In Inflammatory Environment And Its Role In The Pathogenesis Of Osteoarthritis Based On Single-cell RNA Sequencing And Bioinformatics Analysis

Localized inflammation is the causative factor of OA,which occurs earlier than cartilage destruction.The research on the mechanism of OA inflammation has always focused on tissues and cells.It is hard to explore the state of individual cells in the inflammatory environment with high resolution and the details that cells evolved into the stage of irreversible cartilage degeneration which leads to the disease state.OBJECTIVE:To study the heterogeneous response of expanded chondrocytes in vitro in the inflammatory environment induced by interleukin IL-1? at single-cell resolution,to analyze and verify the degree of immune infiltration of the tissues around the joint and the mechanism to drive phenotypic transition of chondrocytes,using of bioinformatics tools to explore the communication situation between chondrocytes in the process of OA and the activation of important transcription factors in the process.METHOD:1.The human-derived articular chondrocytes were expanded in vitro for 13 days.The obtained cells were treated with IL-1? for 0,24,and 48 hours.The processed cells were collected and single-cell RNA sequencing was performed.A variety of bioinformatics tools were used and related experiments were designed to clarify the characteristics of chondrocyte function changes at single-cell resolution.2.A data set containing the expression profiles of meniscus tissues from 12 OA patients and 12 non-OA patients was downloaded,as well as the accompanying clinical information.Xcell was used to evaluate the degree of immune infiltration and its correlation with clinical features,and WGCNA was applied to identify immune-related gene modules.The in vitro molecular biology experiments were carried out to verify immune infiltration and explore the possible mechanism of driving chondrocyte inflammation.3.The sc RNA-seq data set of human chondrocytes in different stages of OA was re-analyzed.The data was re-clustered and annotated through the seurat function package.The ligand-receptor reference database is compared based on the calculation principle of Cellchat and the cell communication network was constructed.The SCENIC method was used to map the gene regulatory network and the AUCell function package was used to score the activity of transcription factors in each cell.RESULT:Single-cell sequencing analysis showed that two major cell clusters with distinct expression patterns were identified at the initial phase and were with heterogeneous variation that coincides with inflammation progress.They transformed into two terminal cell clusters one of which exhibited OA-phenotype and proinflammatory characteristics through two paths,“response-to-inflammation” and “atypical response-to-inflammation”,respectively.The involved cell clusters exhibited intrinsic relationship with cell types within native cartilage from OA patients.Genes controlling cell transformation to OA-phenotype were relating to the tumor necrosis factor(TNF)signaling pathway via NFKB,up-regulated KRAS signaling and the IL2/STAT5 signaling pathway and pathways relating to apoptosis and reactive oxygen species.The different degree of immune infiltration in meniscus between the OA and non-OA groups was detected.The weighted gene co-expression network analysis(WGCNA)identified gene modules closely related to OA and macrophages.The genes in the module were highly enriched in processes related to immune response,such as "antigen processing and presentation","polypeptide antigen assembly of MHC class II protein complexes","antigen processing and peptide or polysaccharide antigen presentation through MHC class II" ","T cell costimulation" and "T cell receptor signaling pathway".The KEGG analysis showed that the pathways were enriched in "antigen processing and display","graft-versus-host disease","allograft rejection","cell adhesion molecules","phagosomes","rheumatoid joints" and "inflammation" and pathways related to a variety of autoimmune diseases.The dominant genes in the module are NCF1,C1 QB,AHR,C1 QC,NCF1B,HLA-DRA,TMEM176 B,CYBB,ITGAM and PTPRC,most of which are known to interact with immunity and inflammation.In vitro experiments also showed that the tissues around the joints have a stronger immune activation and the abnormal reactive reactive oxygen species(ROS)levels under the co-culture with macrophages.The dominant cells in the early progression of OA are effector chondrocytes,which are affected by the external environment.Autocrine and paracrine communication forms were found between cells and the activated 25 signal pathways including BMP pathway and TGF? pathway were predicted in the earlier stage.The dominant cells evolved into pre-hypertrophic chondrocytes,hypertrophic chondrocytes and proliferating chondrocytes during the advanced OA stage.The activated transcription factors in each stage are different while each cluster has the unique activated transcription factor.At the terminal stage,the dominant cell clusters exhibited activated TFs that are obviously related to OA,such as RUNX2,CREB3L1 and TWIST1.However,early activated transcription factors such as FOXA2,BHLHE40,and FOXC2 should be paid more attention.CONCLUSION:This study depicts a high-dimensional transcriptome map of chondrocytes expanded in vitro in an IL-1?-induced inflammatory environment,revealing multiple chondrocyte subpopulations with different functions during the progression of inflammation.The "response-to-inflammation" pathway that continues to evolve toward end-inflammatory-specific chondrocyte subpopulations has been identified.This subpopulation and the genes that determine the evolution to this subpopulation and their expression products may represent a new therapeutic target for OA.The WGCNA has obtained a gene module with 10 closely related to OA and degeneration.Macrophage infiltration and chondrocyte dysfunction,as well as inflammatory factor imbalance and ROS burst may be the cause of early OA inflammation after the immune infiltration of the tissue around the cartilage.The early dominant cells in the progression of OA are effector chondrocytes,which undergo intercellular communication in the form of autocrine and paracrine after being affected by the external environment.The dominant cells evolved into pre HTC,HTC and pro C during the advanced OA stage.The activated transcription factors in each period are not the same,and each cluster has its unique activated transcription factor.