Single-cell and population NF-κB dynamic responses depend on lipopolysaccharide preparation

Area of Science:

• Immunology
• Cellular Biology
• Microbiology

Background:

• Lipopolysaccharide (LPS) from gram-negative bacteria activates Nuclear factor (NF)-κB via Toll-like receptor 4.
• Cellular responses to LPS are known to vary based on ligand source and preparation.
• Understanding these variations is crucial for interpreting experimental results.

Purpose of the Study:

• To compare single-cell Nuclear factor (NF)-κB dynamics across different sources and concentrations of lipopolysaccharide.
• To investigate how variations in LPS preparation affect cellular signaling.
• To determine the impact of LPS source on Toll-like receptor 4 mediated responses.

Main Methods:

• Live-cell fluorescence microscopy was employed to monitor NF-κB activation dynamics in single cells.
• Computational image analysis quantified the nuclear localization of a p65-dsRed fusion protein over time.
• Three distinct Escherichia coli lipopolysaccharide preparations were tested across a wide concentration range.

Main Results:

• Lipopolysaccharide preparations induced markedly different cellular responses, even when accounting for potency variations.
• Single-cell analysis revealed significant heterogeneity in Nuclear factor (NF)-κB activation dynamics.
• The efficacy of soluble TNF receptor in modulating NF-κB dynamics varied strikingly between LPS preparations.

Conclusions:

• Cellular responses to lipopolysaccharide are highly sensitive to the specific source and preparation of the ligand.
• Experimental findings derived from one LPS preparation may not be generalizable.
• Caution is advised when extrapolating results across different lipopolysaccharide preparations.