Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression...

Area of Science:

• Cell Biology
• Immunology
• Optogenetics

Background:

• Toll-like receptor 4 (TLR4) activation by lipopolysaccharide (LPS) in endothelial cells (ECs) triggers pro-inflammatory mediators.
• Systemic release of these mediators contributes to sepsis and chronic inflammatory diseases.
• LPS stimulation lacks precise temporal control and specificity due to off-target interactions.

Purpose of the Study:

• To engineer novel optogenetic endothelial cell lines for precise temporal and reversible activation of TLR4 signaling.
• To compare TLR4 pathway activation induced by light versus LPS stimulation.
• To investigate the functional consequences of optogenetically controlled TLR4 signaling.

Main Methods:

• Development of light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs).
• Quantitative mass spectrometry, RT-qPCR, and Western blot analysis to assess protein and gene expression.
• Functional assays including THP-1 cell chemotaxis, EC monolayer disruption, and transmigration assays.

Main Results:

• Light stimulation of opto-TLR4-LOV cells resulted in distinct pro-inflammatory protein expression profiles and kinetics compared to LPS.
• Light induction promoted THP-1 cell chemotaxis, EC monolayer disruption, and transmigration.
• ECs with a truncated TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) showed high basal activity and rapid signaling depletion upon illumination.

Conclusions:

• The engineered optogenetic cell lines enable rapid and precise photoactivation of TLR4 signaling in endothelial cells.
• This optogenetic approach offers a receptor-specific method for studying TLR4-mediated inflammatory responses.
• The developed tools facilitate detailed investigations into the temporal dynamics of TLR4 signaling pathways.