The Role And Mechanism Of ERAP2 In CD4~+ T Cell Pyroptosis In Rheumatoid Arthritis

Objective:Rheumatoid arthritis(RA)is a chronic and progressive autoimmune disease.Its pathogenesis is complex and has not yet been fully elucidated.T cell pyroptosis is an important pathogenic factor of RA.This study aims to investigate the role of Endoplas-mic Reticulum Aminopeptidase 2(ERAP2)in the pyroptosis of RA CD4~+T cells and its specific molecular mechanism.Methods:Human peripheral venous blood was collected,purified and activated,and cultured for 72 h.The expressions of pyroptosis related proteins and ERAP2 in CD4~+T cells of healthy control(NC)group and RA group were detected by Western Blot.Flow cytometry was used to detect cell death and activated Caspase-1 in both groups.The expression levels of pyroptosis related molecules and ERAP2 in the two groups were detected by PCR.The expression of IL-1βin culture medium was detected by ELISA and the release level of lactate dehydrogenase(LDH)in culture medium was detected by enzyme label.Two lentiviruses,sh ERAP2 and LV-ERAP2,were used to transfect CD4~+T cells in RA group and NC group after purifying human CD4~+T cells and cul-ture for 72 h.The pyroptosis levels of CD4~+T cells in NC-shcontrol,RA-shcontrol,RA-sh ERAP2,LV-NC and LV-ERAP2 groups were determined by flow cytometry,LDH kit,ELISA kit and western blotting.Hoechst 33342/PI fluorescence staining was used to detect PI intake in each group.3 groups of NC-shcontrol,RA-shcontrol and RA-sh ERAP2 CD4~+T cells treated with negative control virus and sh ERAP2 virus were injected by caudal vein 7 days after the synovium of healthy human was implanted sub-cutanetly into the back of NCG mice.After 7 days,the activation of Caspae-1 in resi-dent CD4~+T cells in each group was detected by tissue immunofluorescence technique,and the expression of related inflammatory molecule genes in each group was detected by PCR.Western Blot was used to detect the expression of Hedgehog/SMO/Gli1 axon-related protein in NC group and RA group,and GLI1 antagonist and SMO receptor ag-onist were used to intervene the cells after ERAP2 knockdown and overexpression. Then,the protein expressions of ASC,NLRP3,active Caspase-1 and GSDMD-N in each group were detected.Results:1.The gene and protein expressions of ERAP2 in CD4~+T cells of RA patients were significantly increased,and knockdown or overexpression of ERAP2 could signif-icantly down-regulate or up-regulate the pyroptosis level of CD4~+T cells.2.After ERAP2 knockdown,the expression level of Caspase-1 in CD4~+T cells in the synovial tissue was significantly decreased,and the expressions of pro-inflammatory factors TNFA,IL6 and IL1βwere significantly down-regulated in the membrane tissue,while the expressions of anti-inflammatory molecules IL10 and TGFB1 were significantly in-creased.3.The activation of Hedgehog signaling pathway was significantly down- regulated in RA CD4~+T cells,which showed an opposite trend to the expression of ERAP2,while the activation level of Hedgehog signaling pathway was significantly up-regulated in CD4~+T cells that knocked down ERAP2.Moreover,activation of Hedge-hog signaling pathway attenuates the pyroptosis of RA CD4~+T caused by overexpres-sion of ERAP2.Conclusion:1.ERAP2 is highly expressed in CD4~+T cells of patients with rheumatoid arthritis,and induces pyroptosis of CD4~+T cells.2.ERAP2 regulates the development of rheumatoid arthritis by inhibiting the Hedgehog signaling pathway and promoting the pyroptosis of CD4~+T cells.