Dissection of MAPK and NF-κB p65 signaling dynamics underlying LPS tolerance in macrophages at the single-cell level

MAPK and NF-κB pathways regulate macrophage responses to microbial stimuli. Repeated exposure to lipopolysaccharide (LPS) induces endotoxin tolerance, a state in which inflammatory cytokine production is suppressed while antimicrobial functions are preserved. Although regulatory mechanisms of LPS tolerance is well established, how prior LPS exposure reshapes signaling dynamics downstream of TLR4 remains unclear. Using biosensors and live-cell imaging, we quantified ERK and NF-κB signaling in RAW264.7 macrophages during LPS tolerance. Tolerized macrophages produced less TNF-α and IL-6 and showed reduced ERK and NF-κB activity, with lower amplitudes and areas under the curve than cells receiving a single LPS stimulation. Both pathways also displayed delayed activation, reflected by a prolonged time to first peak. Inhibition experiments revealed bidirectional crosstalk, as blocking ERK altered NF-κB signaling and NF-κB inhibition suppressed ERK dynamics. These findings show that LPS tolerance involves coordinated changes in the strength and timing of ERK and NF-κB signaling.