Decreased ALKBH5 in neutrophil correlates with disease activity in rheumatoid arthritis and ALKBH5 modulates neutrophil autophagy

Neutrophils are key immune components in rheumatoid arthritis (RA), but the role of ALKBH5 in neutrophil function and RA progression remains unclear. METTL3, METTL14, WTAP, ALKBH5, FTO, YTHDF2, autophagy related gene were investigated by RT-qPCR. Autophagy, LC3B were respectively detected by flow cytometry analysis, Western blotting. Correlation analyses between ALKBH5 levels and RA disease activity, autophagy were performed, and receiver operating characteristic (ROC) curves were constructed to evaluate its predictive value. Univariate analysis and multivariate regression analysis were used to analyze the risk factors and construct predictive model. Sh-ALKBH5 lentiviral vectors was constructed and used to infect HL-60 cell line. The role of ALKBH5 in autophagy regulation was explored using RNA immunoprecipitation with next generation sequencing (RIP-Seq), m6A RNA Methylation Analysis, m6A immunocoprecipitation-quantitative polymerase chain reaction (MeRIP-qPCR) and Actinomycin D treatment. Results showed significantly reduced ALKBH5 levels in RA neutrophils compared to healthy controls (HC) and ankylosing spondylitis (AS) patients, correlating with RA disease activity. A novel predictive model incorporating ALKBH5, hemoglobin (HGB), and lymphocyte percentage (L%) exhibited enhanced efficacy in distinguishing RA patients from HC (AUC = 0.937) and AS patients (AUC = 0.943), and could reflect RA severity and activity. Additionally, autophagy levels in neutrophils were highest in RA synovial fluid, followed by RA peripheral blood, and lowest in HC peripheral blood, with ALKBH5 expression showing the opposite trend. Moreover, ALKBH5 negatively correlated with neutrophil autophagy and the silencing of ALKBH5 in HL-60 enhanced the autophagy. Silencing ALKBH5 increased m6A level of ATG7 mRNA and ATG7 mRNA stability. Mechanistically, ALKBH5 inhibited neutrophil autophagy through mediating m6A modification of the ATG7 mRNA. In conclusion, these findings demonstrate that ALKBH5 regulates neutrophil autophagy in RA, and the ALKBH5-HGB-L% model holds potential as a diagnostic and disease activity indicator for RA. Briefly, The HL-60 was treated with cell lysis buffer. Ten percent of the lysate was stored as "input," 80% was used for immunoprecipitation with anti-ALKBH5 antibody (Proteintech, Wuhan, China) ("IP"), and 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control ("IgG").