uORFs identified in this study.

Macrophages require rapid and tightly controlled regulatory mechanisms to respond to environmental disruptions. While transcriptional regulation has been well characterized, the mechanisms underlying translational control in macrophages remain poorly understood. Here, we investigated the dynamics of mRNA translation in mouse macrophages during acute, intermediate, and prolonged LPS exposure. Our results reveal clear phase-specific translational regulation during macrophage polarization, which initially increases the synthesis of inflammatory mediators and cytokines, while simultaneously suppressing the expression of cell cycle-related genes. Mechanistically, we observed pervasive upstream translation in the 5′ UTRs of cell cycle-related mRNAs, which contributes to cell cycle arrest during the early phase of inflammatory response. Notably, we identified a unique codon preference toward A/U in the third position of codons in macrophages, which contrasts with the G/C preference commonly observed in other tissues. AU codon preference increases the stability and translation efficiency of cell cycle-related mRNAs, promoting cell cycle restoration after extended LPS exposure. These findings reveal that uORF translation and codon usage bias are critical components of translational regulation during macrophage polarization, highlighting a potential therapeutic intervention for modulating immune activation via macrophage-specific codon optimization.