Heterogeneity And Lineage Analysis Of Synovial Membrane MSCs And Their Roles In The Pathogenesis Of Rheumatoid Arthritis

Background:Rheumatoid arthritis（RA）is an autoimmune disease characterized by extensive synovium hyperplasia and progressive joint destruction.It can cause high rate of disability and deformity,which seriously endanger national health and affect the survival quality of patient.Excessive proliferation of synovium is the initiating link in RA and one of the main characteristics of RA.Fibroblast-like synoviocytes（FLSs）play a key role in joint inflammation,synovium hyperplasia,cartilage and bone erosion.Our previous research has confirmed that transplantation of exogenous bone marrow or umbilical cord derived mesenchymal stem cells（MSCs）could supress synovium hyperplasia and bone erosion in RA animal models and patients^[1].In recent years,studies have found that MSCs also exist in the synovium（SM-MSC）.SM-MSC have stronger proliferative capacity compared to bone marrow and adipose MSCs,as well as stronger chondrogenic potential compared to umbilical cord MSCs^[4-5].In vitro studies have also shown that it has the ability to inhibit T cell proliferation^[6],therefore,it’s believed to be helpful in maintaining and repairing joint tissue^[7].However,why does the presence of SM-MSCs fail to prevent persistent joint inflammation and progressive bone erosion in RA patients?What is the role of SM-MSCs in the occurrence and development of RA synovitis?Does the synovium hyperplasia originate from the pathological transformation of SM-MSCs?Whether SM-MSCs and FLSs with similar biological characteristicshave are the same cell or different developmental stages of the same cell?What are the key genes that determine the fate of SM-MSCs?Currently,there is no relevant research.Objective:1.To reveal the heterogeneity of synovium tissue in RA and the changes in biological characteristics and functions of SM-MSCs2.To clarify the subgroup changes and differentiation lineage characteristics of SM-MSCs in CIA mice during the development of arthritis,and explore the relationship between SM-MSCs and FLSs.Methods:1.Transcriptome sequencing and analysis of synovium tissue in RA patients group and healthy control groupThe synovium tissues were abtained from RA and healthy control.Meanwhile,HE staining was performed to clarify the pathological and physiological manifestations.Then,transcriptome sequencing was performed to detect the heterogeneity of synovium tissue between the two groups.According to previous literature,selecting heterogeneous genes that may be related to the stemness and differentiation of SM-MSCs.2.Comparison of biological characteristics and functions of SM-MSCs between RA patients group and healthy control groupImmunofluorescence assay was used to detect the specific surface antigen of MSCs such as CD44 and stem genes（OCT4 and SOX2）in the synovium membrane.SM-MSCs were cultured and identified in vitro.Immunofluorescence and PCR were used to detect stem gene expression,CCK8 was used to detect proliferation abilit,Transwell was used to detect invasion ability,scratch test was used to detect migration ability,mixed lymphocyte test was used to detect the immune regulation ability,moreover,staining and RT-q PCR was appliedd to detect the differentiation ability.3.Transcriptome sequencing and analysis of synovium tissues in CIA mice of different stagesDBA/1J mice were randomly divided into normal control groups,early,middle,and late stage groups.The synovium tissue of the hind limbs knee joints was subjected to transcriptome sequencing to clarify the heterogeneity of gene expression in the synovium tissue at each stage of the model.4.Sequencing and analysis of single cell transcriptome in synovium tissues of CIA miceSingle cell sequencing was performed on the synovium tissue in early,middle,and late CIA mice.Firstly,analyzing all cell types in the synovium tissue,and comparing the distribution characteristics of these cell types in mice synovium tissue at each stage.Cluster analysis was performed on FLSs subgroups,annotation was performed on each cellsubgroup according to marker gene expression,and analysis was conducted on the distribution of each subgroup and specific gene expression profile during the occurrence and development of CIA.Finally,through pseudotime analysis,analyzing the FLSs subgroup changes and SM-MSCs lineage differentiation during the formation of RA and identifying the key genes responsible for the pathological transformation of SM-MSCs.5.Key gene validationThe RA patient differential genes were intersected with mice differential genes.Then,they are mapped to the mice synovium tissue single cell sequencing UMAP.The genes enriched in the FLSs subgroup were used as target genes for validation in vitro cultured RA and healthy control SM-MSCs.Results:1.Transcriptome sequencing and analysis of synovium tissue in RA patients and normal controls（1）Synovium histopathology:In the normal control group,there was no hyperplasia of synovium tissue and no significant inflammatory cell infiltration.In RA group,the synovium tissue significantly proliferated,with interstitial osteoporosis and edema,chronic inflammatory cell infiltration,massive neovascularization and cellulose deposition.（2）Cluster analysis and functional enrichment analysis of differential genes between RA patients and healthy controls:using q<0.05 and∣FC∣>1 as the screening condition,a total of 183 differential genes were selected,including 110 up-regulated genes and 73down-regulated genes.The differential gene functions in synovium tissue of RA patients and healthy controls are enriched in extracellular matrix,cell migration regulation,inflammation.（3）Protein-protein interaction network analysisPPI network showed that key proteins include LUM,ACAN,COL2A1,VEGFA,FMOD,CXCL9,COL2A1,MMP13,etc.（4）Target gene heat map analysisThe genes related to stemness,proliferation,invasion,and differentiation of SM-MSCs were used as target genes to compare their gene expression in two synovium groups.The results showed that the expression of stemness gene SOX10 in RA group was lower than that of control group,while the expression of PODNL1,BHLHE22,PERLP,AEBP1,LTBP2,AMTN,NELL1,CLIP2 genes increased in RA group.2.Comparison of biological characteristics and functions of SM-MSCs between RA patients and normal controls（1）In vitro culture and identification of two groups of SM-MSCsIn vitro culture,both groups of SM-MSCs showed long spindle shaped adherent growth.Flow cytometry showed that CD73,CD44,CD29,and CD90 were high expressed,while CD45,CD14,CD34 and HLA-DR were low expressed.Immunofluorescence assay showed that both SOX2 and OCT4 were positive,indicating that the cells were SM-MSCs.（2）Comparison of biological characteristics between two groups of SM-MSCsThe stemness gene detection results of the two groups of SM-MSCs showed that the expression of SOX2 decreased and no significant difference in OCT4 expression..The proliferation,migration,and invasion abilities of SM-MSCs derived from RA patients were higher than those of the control group.（3）Comparison of biological functions between two groups of SM-MSCs（1）The comparison of immune regulatory ability between the two groups showed that SM-MSCs derived from the healthy control group could downregulate the level of TNF-α、IL-17、IFN-γand increase the level of IL-10 in the supernatant of mixed lymphocyte reaction cultured cells.SM-MSCs from RA patients can upregulate TNF-α、IL-17、IFN-γlevel,but the effect on the increase of IL-10 level was weakened.The above differences are statistically significant.At the same time,studies have shown that both groups of SM-MSCs can upregulate the expression of IL-6,especially in the RA group.（2）The differentiation ability test results of the two groups showed that SM-MSCs from both RA and healthy control group could differentiate into osteogenic,adipogenic,and chondrogenic directions under the induction of corresponding induction media.Quantitative comparison of differentiation related genes by PCR showed that compared with the control group,RA derived SM-MSCs expressed more RUNX2 after osteogenesis induction and less ADD1 after lipogenesis induction.3.Detection of synovium tissue heterogeneity in CIA mice at different stages（1）Macroscopic,imaging,and pathological changes in CIA mice at different stagesIn the early CIA group,the joints appeared red and swollen,with limited activity.Histopathology showed infiltration of inflammatory cells in the synovium tissue,with mild synovium tissue hyperplasia.Pathology and X-ray did not show significant cartilage and bone destruction.In the middle stage group,the swelling and pain of the joints reached the peak,with a large amount of synovium tissue proliferation,pannus formation,and significant cartilage and bone erosion.In the late stage group,the redness and swelling of the joints were reduced,the joints were stiff and deformed,a large amount of extracellular matrix was visible in the synovium tissue of the joints,and the normal structure of the joints disappeared.（2）Analysis of synovium tissue heterogeneity between CIA mice and control groups（1）Principal component analysis of synovium membrane samples from four groups of mice:Compared with the control group,there were significant differences among the early,middle,and late CIA groups,with the most significant differences between the mid CIA group and the control group.（2）Analysis of differential genes and functional enrichment between mice in CIA groups and control group:using q<0.05 and fold change>1 as the screening condition,a total of 772 differential genes were selected,including 682 up-regulated genes and 90down-regulated genes.Upregulated genes are enriched in pathways such as complement activation pathway,PI3K-Akt-m TOR signaling pathway,inflammatory response,endochondral osteogenesis,oxidative stress injury,and matrix metalloproteinases.（3）Synovium specific gene analysis of different groups:by crossing the different genes obtained from the synovium tissue of mice in the early,middle,and late CIA groups compared with the control group respectively,137 specific genes in early group,104 specific genes in mid group,and 294 specific genes in late group were obtained.Synovium tissue specific genes in early CIA mice are involved in inhibiting apoptosis and promoting angiogenesis,inflammation related,chemokines and their receptors,regulating cell proliferation,apoptosis,differentiation,transformation,and promoting upregulation of osteoclast differentiation related genes.synovium tissue specific genes in the mid CIA mice group involve upregulation of genes related to immune response,inflammation,memory consolidation,and osteoclast differentiation.Late group specific genes include:downregulation of genes related to promoting pluripotency and stem cell maintenance;upregulation of angiogenesis promotion,collagen formation and assembly,matrix degradation,connective tissue remodeling,and other related genes.（3）Study on the changes of SM-MSCs subpopulations and differentiation lineages during the occurrence and development of synovitis in CIA mice（1）synovium tissue subgroup analysis of mice in each group:This study obtained 8cell types through cluster analysis,including B cells,endothelial cells,FLSs,granulocytes,macrophages,monocytes,smooth muscle cells（SMCs）,and T cells.A separate cluster analysis of 2192 FLSs yielded 7 cell subsets.Each subgroup was annotated based on the expression of the marker gene in each subgroup:FLS1 subgroup-angiogenic phenotype;FLS2 subgroup-invasive phenotype;FLS3 subgroup-immunocyte recruitment phenotype;FLS4 subgroup-inflammatory phenotype;FLS5subgroup-fibrosis phenotype;FLS6 subgroup-an unclassified subgroup,FLS7 subgroup-SM-MSCs phenotype.（2）Distribution of seven FLS subsets in four synovium tissue:The dominant FLS subgroups in the early CIA group are FLS2 and FLS3 subgroups,which mainly involve immune cell recruitment and invasion phenotypes.The dominant subgroups in the mid stage group are FLS1,FLS5,and FLS4,which involve pannus formation,inflammation,and extracellular matrix formation phenotypes.The overall number of FLSs in the normal control group and the late stage group is small,but the expression of stemness related genes is higher than that in the early and mid stage group.（3）SM-MSCs pedigree analysis:A tree diagram of FLSs development trajectory is obtained based on pseudotime analysis.Mapping FLS7（annotated as SM-MSCs subgroup）into the pseudotime series tree diagram showed that it was mainly distributed at the beginning of the tree diagram.Differential gene analysis was performed on each branching subgroup in the tree diagram,and it was found that they expressed chemokine phenotype,angiogenesis promoting phenotype,angiogenesis inhibiting phenotype,fibrosis phenotype and invasion phenotype.（4）Screening and verification of key genes:The differential genes obtained from Part I and Part III were intersected to obtain genes closely related to cell stemness,proliferation,and differentiation.Mapping these genes at the single cell level in mice synovium tissue showed that Fibin,Creb3l1,Chrdl1,Snai1,Glis3,Aebp1,Thbs2,Gpm6b,Clec11a,Twist1,Fosl1,and Medag were highly expressed.RT-PCR was used to detect the expression of the above genes in SM-MSCs in RA patients and healthy controls,whose results showed that there were significant differences in the expression of RUNX2,CREB3L1,THBS2,CLEC11A and TWIST1genes.Conclusion:1.Compared with the healthy control group,the expression of some stem genes in the synovium tissue of RA patients was down regulated,and the expression of functional related genes such as inflammation,chemokines,extracellular matrix formation,bone erosion,and cell migration regulation was up regulated,consistent with the pathological characteristics of synovium tissue.2.The RA SM-MSCs showed increased proliferation,differentiation,migration,and invasion ability,down-regulated stemness gene expression,diminished anti-inflammatory effects and even exhibited a pro-inflammatory phenotype,enhanced osteogenic differentiation,and diminished lipogenic differentiation.3.Different stages of mouse synovium tissue have different gene expression profiles.In the early stage of CIA mice,inflammation,chemokines,and angiogenesis were the main phenotypes of synovium tissue.In the middle stage,anti-inflammatory factors were down-regulated,exhibiting immune response,inflammation,and bone destruction phenotypes.In the late stage,extracellular matrix formation,matrix degradation,and connective tissue remodeling were the main phenotypes.4.FLSs can be divided into angiogenic phenotype,invasive phenotype,immune cells recruitment phenotype,and inflammatory phenotype,fibrosis phenotype and SM-MSCs phenotype.SM-MSCs are located at the beginning of lineage differentiation,and exhibit angiogenesis,inhibition of angiogenesis,immune recruitment,fibrosis,and invasion phenotypes over time.Once the balances between angiogenesis and inhibition of angiogenesis,matrix production and degradation are broken,changes in each dominant subgroup occur,which mediates the occurrence and development of synovitis.Aberrant expression of RA RUNX2,CREB3L1,THBS2,CLEC11A,and TWIST1 play an important role in the pathological transformation of SM-MSCs.