PI16 Was Involved In The Pathogenesis Of RA By Regulating The Number And Function Of CD4⁺CD25⁺Foxp3⁺ Regulatory T Cells Via Dot1-L

ObjectiveRheumatoid Arthritis（RA）is a chronic systemic autoimmune disease characterized with synovitis and angiogenesis.Immunological abnormalities in RA include the imbalance and abnormal activation of immune cells.In 1995,Sakaguchi etc firstly found that Regulatory T cells（Treg）play an important role in maintaining immune tolerance and preventing autoimmune reaction in the research of autoimmune disease in mice.In recent years,many studies shown the reduced numbers and dysfuction of Treg cells in RA patients,indicated Treg cells play an important role in the pathogenesis of RA,but the mechanism is unclear.Peptidase inhibitor 16 belongs to the Cysteine Rich Secretory Proteins（CRISP）family,is composed of 436 amino acids,n-terminal have three glycosylation modification,also called CRISP-9 or Prostate Secretory protein binding protein（PSPBP）,which is mainly distributed in heart,prostate,etc.Previous reports found that PI16 was expressed in over 80%of human CD25⁺FOXP3⁺Treg using whole-genome chromatin immunocoprecipitation sequencing analysis,and PI16⁺CD25⁺was considered as one of the molecular markers of Treg.Decreased PI16⁺Treg cells were found in peripheral blood of juvenile idiopathic arthritis patients.The relationship between PI16 and Treg cells and their role in RA are unclear.This research aim to investigate the relationship between PI16 and Treg cells,reveal their potential role in the pathogenesis of RA and further elucidate the molecular mechanism of immune disorders through constructing high-expression PI16 transgenic mice and a series of vivo and vitro experiments.Methods1.Effects of PI16 on the development of immune cells in mice.Previous studies have found that knockouted PI16 can lead to dysontogenosis.PI16 transgenic mouse（PI16Tg）was successfully constructed using the extensive expression promoter EF1-α.The high expression of PI16 was conformed in spleen,lymph node,thymus and myocardium of PI16 transgenic mouse.Flow cytometry was used to detect the expression of immune cells（T,B celland neutrophil）in the spleen of PI16Tg mice at 6 weeks and 8 weeks.2.Effects of PI16 on the expression of CD4⁺T subsets and Treg cells in the spleen of mice.Detected the propotions of Th1,Th2,Th17,Tfh and Treg cells in the spleen of PI16 transgenic mice and WT mice at 10 weeks by Flow cytometry.Compared the propotions of n Tregs and i Tregs in spleen,thymus and lymph nodes between PI16transgenic mice and WT mice at 10 weeks by Flow cytometry.3.Effects of PI16 on the differentiation,proliferation and apoptosis of Treg cells.Separated CD4⁺CD25^-T cells in the spleen from PI16 transgenic mice and WT mice by magnetic beads and induced it to differentiation into Treg cells in vitro,then observed the effects of PI16 on the differentiation,cell proliferation and apoptosis of Treg cells.4.Effects of PI16 on the inhibitory function of Treg cells.Separated Treg,Teff and APC cells in the spleen from PI16 transgenic mice and WT mice by magnetic beads.Lymphocyte co-culture system was used to observe the effect on Treg inhibitory function.5.Effects of PI16 on the expressions of related genes of Treg cells.RT-PCR was used to detect the expression of related genes（Foxp3,CTLA-4,IL-10,TGF-β）of Treg cells in the spleen from PI16 transgenic mice and WT mice.6.Effects of Dot1-L on the differentiation of Treg cells in PI16Tg mice.Ch-IP analysis revealed that PI16 can specifically bind to Dot1-L in cardiomyocytes.The levels of Dot1-L in the spleen of PI16 transgenic mice and WT mice was detected using RT-PCR.The interaction between Dot1-L and PI16 and the possible binding sites was investigated via Duo-link PLA.Separated CD4⁺CD25^-T cells in the spleen from PI16 transgenic mice and WT mice by magnetic beads and induced it to differentiation into Treg cells in vitro,then cultured with or without EPZ-5676,and observed the proportion of Treg cells.7.Effects of PI16 on the pathogenesis of collagen induced arthritis（CIA）.Type II collagen was used to induce arthritis（CIA）in PI16Tg mice and WT mice（background:C57BL/6）.Histopathology was used to observe the effects of local inflammation and the number of osteoclast in joints of the CIA mice.8.The expression of PI16 in RA patients and CIA mice.To verify the relationship between P116 and the disease,the immunohistochemistry,ELISA,RT-PCR and flow cytometry was used to detect the expression of PI16 in the synovium,serum,PBMC and T cells from RA patients,the analyzed the correlation between PI16⁺CD4⁺cells and Th17,Treg,Th17/Treg,ESR.Moreover,the changes in the expression of PI16 and Treg-related genes in synovium and spleen at different time points of onset were further analyzed in the CIA model of DBA/1J mice by RT-PCRResults1.Effects of PI16 on the development of immune cells in mice.The results of Flow cytmetry shown that,at 6 weeks,the propotion of T cells（25.40±1.47%vs.22.23±1.59%,p=0.2181）,B cells（60.93±3.95%vs.61.20±2.58%,p=0.9577）and neutrophils（9.01±1.49%vs.9.05±0.98%,p=0.9826）in the spleen had no obvious difference between PI16Tg mice and WT mice.At 8 weeks,compared with WT mice,the propotion of T cells（25.13±0.37%vs.20.43±1.02%,p=0.0122）in the spleen of PI16Tg mice was significantly increased,while the propotion of B cells（24.87±1.51%vs.29.00±6.154%,p=0.5498）and neutrophils（15.73±3.53%vs.10.67±1.62%,p=0.2452）had no obvious difference.2.Effects of PI16 on the expression of CD4⁺T subsets and Treg cells in the spleen of mice.The results of Flow cytmetry shown the proportion of Treg cells（5.66±0.52%vs.8.52±0.44%,p=0.0028）was significantly reduced in the spleen of PI16transgenic mice,the percentage of Th1（9.60±0.89%vs.9.88±1.71%,p=0.8914）,Th2（3.24±0.75%vs.3.42±0.99%,p=0.8923）,Th17（4.38±0.63%vs.4.98±1.19%,p=0.6522）,Tfh cells（1.46±0.42%vs.2.46±0.66%,p=0.2679）had no obvious difference.The percentage of n Tregs（5.05±0.22%vs.6.93±0.44%,p=0.0187）in the spleen of PI16 transgenic mice was significantly reduced at 10 weeks.,the percentage of NTregs and i Tregs had no significant change in thymus and lymph nodes.3.Effects of PI16 on the differentiation,proliferation and apoptosis of Treg cells.Compared to WT mice,the differentiation ratio of Treg cells induced from CD4⁺CD25^-T cells of PI16Tg mice was lower（56.96±0.83%vs.66.55±0.64%,p=0.0008）),and the proliferation index of Treg cells from PI16Tg mice（1.63±0.66）was significantly lower than those from WT mice（2.39±0.21,p=0.0245）.There was no significant difference in the apoptosis of Treg cells（2.26±0.12%vs.1.79±0.32%,p=0.2333）.4.Effects of PI16 on the inhibitory function of Treg cells.The Treg inhibitory function experiment shown that the cell proliferation index of Teff cells was higher in the presence of Treg cells from the PI16 transgenic mice（2.23±0.07）than the Treg cells from the WT mice（1.52±0.06,p=0.0158）.Both of two groups were lower than the Control group（4.77±0.04,p<0.05）.The inhibition ability of Treg cells from the PI16 transgenic mice decreased.5.Effects of PI16 on the expression of related gene of TregThe results of RT-PCR shown no significant difference in the expression of Foxp3,CTLA-4,IL-10 and TGF-βin the spleen of PI16 transgenic mice and WT mice.6.Effects of Dot1-L on the differentiation of Treg cells in PI16Tg miceDot1-L was higher expressed in the spleen of PI16 transgenic mice than WT mice.Duo-link assay show that PI16 on Treg cells can directly interact with Dot1-L proteins.Futher in vitro differentiation experiment shown the lower differention ratio of Treg cells in the PI16 transgenic mice group（51.13±3.657%,）could be reversed by EPZ-5676（61.47±0.5517%vs.51.13±3.657%,p=0.0313）.7.Effects of PI16 on the pathogenesis of collagen induced arthritis（CIA）.（1）Osteoarthritis score:compare to WT mice,PI16 transgenic mice had a higher incidence,earlyer onset,and more severe onset,with longer duration.（2）Pathological changes of the joints:HE staining shown the damaged of the joint structure was more severer in PI16 transgenic mice than WT mice,and inflammatory cell infiltration was more obvious.8.The expression of PI16 in RA patients and CIA miceImmunohistochemical results confirmed elevated expression of PI16 in RA synovial tissues than HC,ELISA results shown the increased level of PI16 in RA patients serum（170.70±11.80 ng/ml,n=43）than HC（93.77±4.89 ng/ml,n=27）（p<0.0001）,RT-PCR shown that PI16 m RNA expression was higher in the PBMC of RA patients（p<0.0001）.In addition,the expression of PI16⁺CD4⁺T cells was higher in RA patients（17.43±1.57%,n=20）than HC（12.50±0.92%,n=10,p=0.0436）.There was no significant correlation between PI16 level and Treg cell,Th17 cell,Th17/Treg ratio and ESR in RA patients..In CIA model,the expression of PI16 gene in the synovial membrane and spleen were gradually increased,up to highest at the peak of onset,and the level of Treg-related genes-Foxp3,CTLA-4 and IL-10 were decreased with the aggravation of arthritis,reached the lowest at 35d and return to normal level during the remission.There was no obvious change in the expression of TGF-β.Conclusion1.PI16 was highly expressed both in PBMC,serum and synovial tissue of RA patients and the spleen and synovial tissue of CIA mice.PI16 could promote the the pathological process of CIA.2.PI16 may regulate the number and function of Treg cells partly by Dot1-L to participate in the pathogenesis of RA.