Objective To investigate the role and the underlying mechanisms of N⁶-methyladenosine (m⁶A) reader YTHDF3 in macrophages activation.

Methods shRNA-mediated Ythdf3 knockdown in RAW264.7 cells was performed and these RAW264.7 cells were stimulated with LPS. Then, changes in the pro-inflammatory and anti-tumor functions, including cytokine production, phagocytosis, and tumoricidal ability were evaluated. The effect of Ythdf3 knockdown on the activation of the Toll-like receptor 4 (TLR4) downstream MAPK and NF-κB pathways was assessed by immunoblotting. After Ythdf3 knockdown, the expression levels and mRNA stability of key junction proteins and signaling molecules of the TLR4 signaling pathway were analyzed to identify YTHDF3 target genes and investigate the underlying regulatory mechanism.

Results After LPS stimulation of wild-type RAW264.7 cells, the level of pro-inflammatory factors increased and then decreased. However, the level of YTHDF3 showed the opposite trend to that of pro-inflammatory factors, suggesting that YTHDF3 might play a role in the negative regulation of macrophage activation. shRNA-mediated Ythdf3 knockdown in RAW264.7 cells significantly increased the expression of pro-inflammatory factors, nitric oxide (NO) production, and phagocytosis. In addition, Ythdf3 knocked-down RAW264.7 cells co-cultured with tumor cells exhibited enhanced tumor killing ability. The findings suggested that YTHDF3 deletion could promote LPS-induced activation of RAW264.7 cells and enhance their production of pro-inflammatory factors and tumor killing function. further investigation into the underlying mechanisms revealed that Ythdf3 knockdown inhibited the degradation of Cd36, Irak1, Tab1/2, and Tirap mRNAs, which were key junction proteins and signaling molecules in the TLR4 pathway, which in turn, enhanced the phosphorylation of p38, a downstream key kinase and the activation of macrophages.

Conclusion By targeting the mRNA of the key junction proteins and signaling molecules of the TLR4 pathway, YTHDF3 accelerates their rapid degradation and suppresses macrophage activation. Ythdf3 knockdown significantly promotes macrophage activation and enhances the tumor killing activities of macrophages.