The Mechanism Of PI16 In The Pathogenesis Of Rheumatoid Arthritis By Regulating Foxp3 Expression Via Inhibiting The Ubiquitin Degradation Of Bmi-1

Objective:Rheumatoid arthritis（RA）is one of the autoimmune diseases with high morbidity and disability rate.Although the pathogenesis of RA is still unclear,the breakdown of immune tolerance is considered to be an important part of its pathological process.Regulatory T cells（Treg）play an important role in maintaining immune tolerance.As a key transcription factor of Treg cells,Foxp3 plays an important role in maintaining the development,differentiation and immunosuppressive function of Treg cells.The stable expression of Foxp3 is a key molecule for maintaining immune tolerance.A large number of studies have shown that abnormal function and quantity of Treg increase in RA.In the inflammatory situation of RA,some Treg will lose the expression of Foxp3and even transform into pathological Th17 cells.The mechanism is still unclear.Peptidase Inhibitor 16（PI16）is a 94-amino acid rich protein of the CAP family,which is expressed in more than 80%of human CD25⁺Foxp3⁺Treg cells,and its specific function is poorly understood.Our previous studies have shown that Peripheral blood mononuclear cells（PBMC）,synovial tissue,serum in patients with RA and spleen and synovial tissue of Collagen induced arthritis（CIA）mice have high expression of PI16.The purpose of this study was to clarify the influence of PI16 on the progression of RA and the mechanism of Foxp3 expression in Treg.Methods:1.Effects of PI16 on disease progression and Foxp3 expression in AIA miceEstablishing Antigen-induced arthritis（AIA）to observe synovial hyperplasia and inflammatory infiltration in the knee joint of AIA mice by HE staining.Western blot and immunohistochemical staining were used to detect the expression of inflammatory cytokines in overexpressed PI16 transgenic(PI16^Tg)arthritis mice.Bone damage of AIA model was analyzed by SO-FG and T-COL staining.Flow cytometry was used to detect the proportion of Treg cells and Th17 cells in spleen lymphocytes of AIA mice.The expression of Foxp3 in Treg cells and knee tissues of AIA mice was detected by Western Blot.2.Influence of PI16 on Bmi-1 expression and enrichment of Foxp3 promoter（1）Detecting the expression of Bmi-1 in AIA model mice knee joint and Treg cells by Western Blot,The expression of Bmi-1 in synovial tissue of AIA mice was detected by immunohistochemical staining.Chromatin immunoprecipitation（Ch IP）technique was used to detect the enrichment of Bmi-1 and histone in the Foxp3 promoter.293T cells overexpressed Bmi-1 and（or）PI16,and the interaction between Bmi-1 and PI16was investigated by cellular immunofluorescence and co-immunoprecipitation（Co-IP）.（2）Analyzing the structural domains of PI16 and Bmi-1 and constructing plasmids of PI16 and Bmi-1.Two amino acid sequences of PI16 were truncated into 1-168 and169-436,while three amino acid sequences of Bmi-1 were 1-95,96-228 and 229-326.The plasmids with different truncations were overexpressed in 293T cells,and the specific domain of the interaction between PI16 and Bmi-1 was identified by Co-IP.（3）AIA model mice were intraperitoneally injected with PTC-209（50mg/kg）for consecutive 7 days,and synovial hyperplasia and inflammatory infiltration in the knee joint area of AIA mice were observed by HE staining.Western blot was used to detect the expression of inflammatory factors in AIA arthritis mice.Foxp3 expression in AIA arthritis mice was detected by immunohistochemical staining.The Bmi-1 and histone enrichment levels of the Foxp3 promoter were detected by Ch IP.3.The effect of PI16 on the degradation of Bmi-1 ubiquitinationHis-Bmi-1 and Flag-PI16 were overexpressed in 293T cells,and detecting the half-life of His-Bmi-1.The overexpression of His-Bmi-1 and/or Flag-PI16,Ub,Ub-K48 and Ub-K63 in 293T cells confirmed the effect of PI16 on the degradation of Bmi-1 ubiquitin.Constructing Bmi-1 lysine site mutants to identify the specific sites where PI16 affected the degradation of Bmi-1 ubiquitination.Results:1.Compared with wild-type mouse（WT）arthritis mice,PI16^Tg arthritis mice showed significant inflammation,synovial hyperplasia,and destruction of articular cartilage.Moreover,the expression of Foxp3 decreased in spleen lymphocytes and knee joints of PI16^Tg arthritis mice,and the proportion of Treg cells decreased.2.In PI16^Tg arthritis mice,Tregs and Bmi-1 of PRC1 molecule were significantly increased,which promoted inhibitory histone modification of H3K27me3 and H2A-K119Ub.PI16 can interact with the 1-95 region of Bmi-1 through the 169-436 region.The application of PTC-209,a small molecule inhibitor of Bmi-1,slowed synovial hyperplasia and inflammatory cytokine expression in the knee joint region of AIA mice,and reduced the enrichment of Bmi-1 and inhibitory histone in the Foxp3 promoter region.3.PI16 prolonged the half-life of Bmi-1 and inhibited the degradation of K48ubiquitination at Lysine 73 and 153 sites of Bmi-1.Conclusion:PI16 inhibits Foxp3 expression and promotes disease progression in arthritis mice by inhibiting the degradation of K48-linked polyubiquitination at sites K73 and K153 of Bmi-1 and regulating histone modification in the Foxp3 promoter region.