Transcriptomic profiling reveals transcriptomic remodeling linked to inflammatory activation and cell cycle-related gene expression in the early hours of macrophage activation.

Macrophages undergo rapid transcriptional reprogramming upon LPS stimulation, but the early regulatory mechanisms (≤6 hours) remain poorly understood. This study investigates the immediate molecular responses in the RAW264.7 murine macrophage cell line, focusing on the interplay between immune activation, cell cycle modulation, and metabolic-epigenetic crosstalk. The metabolic and epigenetic crosstalk mentioned in this study is only inferred from transcriptomic data, and no direct experimental verification was performed. Transcriptomic profiling (RNA-seq) was performed on LPS-stimulated (6-hour) and control macrophages. Differentially expressed genes (DEGs) were analyzed via GO/KEGG enrichment and protein-protein interaction (PPI) networks. Key findings were validated by qPCR and Western blot. Identified 2,715 DEGs (716 upregulated, 1,999 downregulated), with Ikbke identified as a multi-pathway gene (14 pathways). LPS triggered activation of inflammatory pathways (NF-κB, TNF) and downregulation of cell cycle regulators. Ikbke and C5ar1 co-enriched in COVID-19 and viral infection pathways, reflecting their involvement in general innate immune signaling pathways. Transcriptomic findings were validated by qPCR and Western blot, confirming a 6.2-fold induction of Ikbke and significant downregulation of Ezh2. This study identifies Ikbke as a potential correlational candidate of early macrophage responses, linking TLR signaling, metabolic shifts, and viral defense mechanisms. All conclusions in this study are limited to the RAW264.7 immortalized murine macrophage cell line and require further verification in primary cells and in vivo models. These findings in the RAW264.7 cell model provide potential molecular targets for further investigating the modulation of early hyperinflammatory responses.