METTL3 contributes to M.tb-induced injury and inflammation in THP-1 macrophages by mediating m6A methylation of IRF8 to activate TLR4/NF-kB pathway.

BACKGROUND

Macrophages play central roles in the immunity response to infection of intracellular bacteria, including Mycobacterium tuberculosis (M.tb) in tuberculosis (TB). Methyltransferase-like 3 (METTL3) has been implicated in the macrophage regulation in TB, and this study intended to investigate the molecular mechanism of METTL3 with interferon regulatory factor-8 (IRF8) in TB using in vitro model established by M.tb-infected THP-1 macrophages.

METHODS

RT-qPCR and Western blot were utilized to analyze mRNA and protein expression, respectively. Cell viability, proliferation, and apoptosis were examined through cell counting kit-8 assay, EdU assay, and flow cytometry/TUNEL assay. Inflammatory cytokines were detected via enzyme-linked immunosorbent assay. Methylated RNA Immunoprecipitation (MeRIP), RIP and Co-IP were performed to assess the interaction between genes.

RESULTS

IRF8 knockdown alleviated injury and inflammation in M.tb-infected THP-1 macrophages. METTL3 enhanced IRF8 mRNA stability by inducing mA methylation. IGF2BP1 functioned as an mA reader to affect mA methylation of IRF8. The function of METTL3 in M.tb-induced THP-1 macrophages was attributed to the positive regulation of IRF8. IRF8 bound to TLR4 and METTL3 could regulate TLR4 expression via targeting IRF8. IRF8/TLR4 axis promoted M.tb-induced THP-1 cell injury and inflammation. TLR4/NF-kB pathway was activated by METTL3-mediated IRF8.

CONCLUSION

These findings revealed that METTL3 expedited cell injury and inflammatory reaction in M.tb-infected THP-1 macrophages by inducing mA methylation of IRF8 to activate TLR4/NF-kB pathway.