Regnase-1 regulates inflammation in T cells of ankylosing spondylitis through the TRAF6

The study aimed to investigate the regulatory role of Regnase-1 in ankylosing spondylitis (AS) inflammation. We collected 10 ml peripheral venous blood and epidemiological data from 45 AS patients and 45 healthy controls and performed enzyme-linked immunosorbent assay (ELISA) experiments to measure the levels of inflammatory cytokines. Then CD3 + T lymphocytes were isolated by magnetic bead sorting method, and the transcriptional levels of Regnase-1 and TNF receptor-associated factor 6 (TRAF6) were detected by real-time quantitative PCR (qRT-PCR). The regnase-1 knockdown human T lymphocyte leukemia cell (Jurkat T) model was constructed by small interfering RNA (siRNA) technology. Then PCR and Western blot were used to detect the transcription level and protein level of downstream genes. Co-immunoprecipitation was used to verify the interaction between Regnase-1 and TRAF6. Regnase-1 and TRAF6 transcription levels were down-regulated and positively correlated with each other in T cells from AS patients. The ROC curve analysis indicates that both Regnase-1 and TRAF6 possess diagnostic capabilities, with Regnase-1 demonstrating a particularly high area under the curve (AUC) of 0.876 (95% CI: 0.789-0.936). Subgroup analysis shows NSAIDs boost Regnase-1 and TRAF6 transcription while reducing IL23 and IL17 levels. The results of cell experiments showed that si-Regnase-1 significantly reduced the mRNA and protein levels of TRAF6 in Jurkat T cells and increased the expression level of inflammatory gene TNF-alpha. Co-immunoprecipitation assay further verified the binding between the two proteins. Regnase-1 may participate in the chronic inflammatory process of AS by regulating TNF-alpha through TRAF6.