Dual-site CRISPR/Cas9-mediated mutations in Toll-like receptor 4 (TLR4): complete characterization reveals divergent autophagic reprogramming of LPS-related downstream activities

This is the authors' abstract. We don't add key points for this paper.

) within TLR4 loci, leading to partial signal propagation and altered LPS trafficking to lysosomal compartments through autophagosome sequestration. Immunofluorescence profiling revealed broad attenuation of TLR-signaling networks, MyD88/TRAF6 recruitment, and downstream NFκB-MAPK-PI3K-AKT-JAK-STAT-mTOR cascades alongside decreased CD14 expression, suppressed ROS generation, and diminished caspase-3 activity. While complementary NFκB and LC3-HiBiT reporter assays confirmed interrupted LPS-induced inflammatory transcription and conventional autophagic flux activation, TFEB reporters revealed that edited macrophages remain highly sensitive and responsive to direct metabolic mTOR-dependent metabolic inhibition via Rapamycin. Collectively, our findings establish TLR4 as a central molecular switch and suggest that CRISPR-Cas9-mediated dual-site editing reprograms macrophages into repair-oriented, adaptive phenotype with implications for therapeutic strategies targeting inflammation, sepsis, and autophagy-driven tissue protection.