Fig 2: ERAP2 induces pyroptosis in RA CD4 + T cells by inhibiting the Hedgehog signaling pathway. CD4+ T cells were stimulated for 72 h. Then, normal CD4+ T cells were infected with control lentivirus or lentivirus encoding ERAP2 (LV-NC/ LV-ERAP2). RA CD4 + T cells were infected with control shRNA virus or ERAP2 shRNA virus (RA-shcontrol/ RA-shERAP2). A, B SHH, SMO, and GLI1 expression in isolated CD4 + T cells is shown in representative immunoblots (left) and plots of the relative band density for NCs (n = 3) and RA patients (n = 3) with normalization to β-actin expression (right). C, D ERAP2, SHH, SMO, and GLI1 expression in isolated CD4 + T cells with overexpression or knockdown is shown in representative immunoblots (left) and plots of the relative band density with normalization to β-actin expression (right). E, F GLI1, ASC, NLRP3, cleaved Caspase-1 and GSDMD-N expression in isolated CD4 + T cells with GANT58 treatment (20 µm) is shown in representative immunoblots (left) and plots of the relative band density with normalization to β-actin expression (right). G, H GLI1, ASC, NLRP3, cleaved Caspase-1 and GSDMD-N expression in isolated CD4 + T cells with Purmorphamine treatment (20 µm) is shown in representative immunoblots (left) and plots of the relative band density with normalization to β-actin expression (right). All data are shown as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple-comparisons test for multigroup comparisons or Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis

Fig 3: ERAP2 regulates pyroptosis in CD4+ T cells. NC and RA CD4+ T cells were stimulated for 72 h. Then, NC CD4+ T cells were infected with control shRNA virus (NC-shcontrol), and RA CD4+ T cells were infected with control shRNA virus or ERAP2 shRNA virus (RA-shcontrol/ RA-shERAP2). A, B ERAP2 expression in isolated CD4+ T cells is shown in representative immunoblots (left) and plots of the relative band density for NCs (n = 5) and RA patients (n = 5) with normalization to β-actin expression (right). C ERAP2 transcript levels in NC and RA CD4+ T cells (n = 5). D, E Flow cytometric analysis of CD4+ T cells treated as indicated and stained with Annexin V/7AAD (n = 3). Representative flow plots of Annexin V and 7AAD staining of CD4+ T cells and a histogram of the percentage of Annexin V+ 7AAD+ CD4+ T cells are shown, and representative scatterplots of FLICA+ cells identified by flow cytometric analysis (n = 3). F Concentrations of secreted IL-1β measured by ELISAs (n = 5). G, H Pyroptotic cells were identified by Hoechst 33,342/PI staining; the nuclei were stained blue with Hoechst 33,342, while pyroptotic cells were stained red with PI (n = 3). Scale bars: 50 μm. I T-cell death was quantified by measuring LDH release (n = 5). J Representative immunoblots showing ASC, NLRP3, cleaved Caspase-1, and GSDMD-N expression (n = 3). K Quantification of ASC, NLRP3, cleaved Caspase-1, and GSDMD-N protein expression shown in J (n = 3). All data are shown as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple-comparisons test for multigroup comparisons or Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis

Fig 4: Overexpression of ERAP2 promotes CD4+ T cell pyroptosis. Normal CD4+ T cells were stimulated for 72 h. Then, CD4+ T cells were infected with control lentivirus or lentivirus encoding ERAP2 (LV-NC/ LV-ERAP2). A Flow cytometric analysis of CD4+ T cells treated as indicated and stained with Annexin V/7AAD (n = 3). Representative flow plots of Annexin V and 7-AAD staining of CD4+T cells and a histogram of the percentage of Annexin V+ 7AAD+ CD4+T cells are shown. B Concentrations of secreted IL-1β measured by ELISAs (n = 5). C Representative scatterplots of FLICA+ cells identified by flow cytometric analysis (n = 3). D T-cell death was quantified by measuring LDH release (n = 5). E Pyroptotic cells were identified by Hoechst 33,342/PI staining; the nuclei were stained blue with Hoechst 33,342, while pyroptotic cells were stained red with PI (n = 3). Scale bars: 50 μm. F Representative immunoblots showing ASC, NLRP3, cleaved Caspase-1, and GSDMD-N expression (n = 3). G Quantification of ASC, NLRP3, cleaved Caspase-1, and GSDMD-N protein expression shown in F (n = 3). All data are presented as the mean ± SD. Statistical significance was determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis

Fig 5: ERAP1 is transcriptionally regulated by p53 via a bona fide p53RE.(a) Real-time qPCR validation of fold change in ERAP1 mRNA expression in HCT116 (p53−/−) cells transfected with wild-type p53 (p53WT) or one of 6 p53 mutants, compared with cells transfected with the pcDNA3.1 control. (b) Real-time qPCR analysis of relative basal ERAP1 mRNA expression levels in p53+/+ and p53−/− HCT116 cells. **P<0.01 (two-tailed Student’s t-test). (c) Western blot of ERAP1, ERAP2, p21, p53 and β-actin (loading control) in HCT116 (p53+/+) and HCT116 (p53−/−) cells. (d) Dual-luciferase assay results of p53WT or the p53 dominant-negative mutant R175H co-transfected with different ERAP1 RE constructs (pGL3-pro-ERAP1-RE1 and RE2, sequences listed in the table at the bottom panel) into HCT116 (p53−/−) cells. Data are presented as percentage luciferase activity relative to the pcDNA3.1 control vector-transfected cells. Luciferase assay using p21 promoter construct (pGL3-pro-p21) serves as the positive control. (e) Binding affinity of wild-type p53 (p53WT) or the six p53 mutants to the identified ERAP1 p53RE sequences (wild-type or mutant) was determined by ProLabel Protein–DNA binding assay. (f) Schematic representation of identified ERAP1 p53RE (RE2) in relation to the ChIP-seq peaks and genomic localization of ERAP1 gene. TSS: transcription start site. Data are representative of three independent experiments and error bars represent s.d. of technical replicates (mean±s.d.), n=3, in a,b,d,e.