Dual-site CRISPR/Cas9-mediated mutations in Toll-like receptor 4 (TLR4): complete characterization reveals divergent autophagic reprogramming of LPS-related downstream activities.
Source: PubMed, NCBI / U.S. National Library of Medicine
Lipopolysaccharide (LPS) initiates hyperinflammatory cascade via the LBP-CD14-MD2-TLR4 complex which can precipitate fatal cytokine storms and septic shock. We aim to develop molecular strategies that selectively dampen TLR4-driven inflammation without eliminating host defense. Here, we demonstrate that CRISPR-Cas9-mediated dual-site editing of the TLR4 gene, by introducing specific disruptions in both the extracellular domain (ECD) and the intracellular Toll/IL-1 receptor (TIR) domain, can generate a partially uncoupled signaling phenotype that selectively attenuates acute inflammation while preserving baseline stress-adaptive mechanisms. By combining whole-genome and amplicon-based next-generation sequencing, RNA-Seq, molecular dynamics simulations, transmission electron microscopy (TEM), GSEA, and functional reporter assays, we show that targeted mutations near the LPS-binding interface within the LRR modules of the ECD (M209I, V254I, E593D) and within the CD loop of the TIR domain (R761H) reduce ligand binding-pocket volume by ~17% (688.19Åvs. 825.41Å) 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.
Abstract
Lipopolysaccharide (LPS) initiates hyperinflammatory cascade via the LBP-CD14-MD2-TLR4 complex which can precipitate fatal cytokine storms and septic shock. We aim to develop molecular strategies that selectively dampen TLR4-driven inflammation without eliminating host defense. Here, we demonstrate that CRISPR-Cas9-mediated dual-site editing of the TLR4 gene, by introducing specific disruptions in both the extracellular domain (ECD) and the intracellular Toll/IL-1 receptor (TIR) domain, can generate a partially uncoupled signaling phenotype that selectively attenuates acute inflammation while preserving baseline stress-adaptive mechanisms. By combining whole-genome and amplicon-based next-generation sequencing, RNA-Seq, molecular dynamics simulations, transmission electron microscopy (TEM), GSEA, and functional reporter assays, we show that targeted mutations near the LPS-binding interface within the LRR modules of the ECD (M209I, V254I, E593D) and within the CD loop of the TIR domain (R761H) reduce ligand binding-pocket volume by ~17% (688.19Åvs. 825.41Å) 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.