Dynamics of ERK/MAPK and NF-κB signaling pathways in macrophages with repeated stimulation by lipopolysaccharide at a single cell level

Abstract

ERK and NF-κB are key regulators of macrophage responses to external stimuli, resulting in the secretion of inflammatory cytokines and exhibiting antimicrobial functions. Macrophage tolerance is a dampened response to repeated lipopolysaccharide (LPS) stimulation and is linked to immunosuppression in patients with sepsis. The dynamics of these two signaling pathways have not been investigated in the context of LPS tolerance. In this study, we utilized live-cell imaging and biosensor technology to assess the dynamics of these two pathways in LPS tolerance using a macrophage cell line stably expressing multiple protein reporters for ERK and NF-κB. RAW264.7, a murine macrophage cell line, was generated to express ERK-KTR Clover fluorescent, NF-κB p65 mRuby2, and H2B Halo nuclei marks for tracking, which is referred to as RAW264.7 Trio. To track the LPS response, cells were stimulated with LPS at 100 ng/ml, then rested for 24 hours before being restimulated with LPS at 10 ng/ml. Cells with repeated LPS stimulation showed dampened IL-6 and TNF-α secretion. The signaling dynamics reveal single-cell heterogeneity, with NF-κB and ERK activity observed in more non-responder cells than in single LPS. Moreover, the signal dynamics showed that LPS-tolerized cells exhibited significantly lower ERK and NF-κB activity compared with the response to 10 ng/ml LPS, as reflected in three signaling features: the area under the curve (AUC), amplitude, and the time to first peak. Taken together, LPS tolerance resulted in decreased signaling dynamics in the ERK and NF-κB pathways and in a shift in the timing of the first response. To elucidate the signaling dynamic crosstalk between ERK and NF-κB pathways during LPS tolerance, specific inhibitors to block ERK or NF-κB signaling were applied 1 hour before LPS restimulation, then monitored signaling dynamics. Inhibiting ERK signaling shifted NF-κB signaling from two peaks to one, while inhibiting the NF-κB pathway suppressed ERK signaling dynamics. This result revealed a bidirectional crosstalk, as blocking ERK altered NF-κB signaling and NF-κB inhibition suppressed ERK dynamics. Insights from this study will enhance our understanding of macrophage responses to LPS tolerance, which may contribute to interventions to alleviate or enhance LPS tolerance in disease settings such as sepsis.