In vitro protocols for inducing and assessing endotoxin tolerance in human and murine macrophages.

Macrophages are key innate immune effector cells capable of responding to microbial factors such as lipopolysaccharide (LPS) through activation of inflammatory pathways. However, repeated LPS exposure can lead to endotoxin tolerance, a reprogrammed state where macrophages suppress pro-inflammatory cytokine production while preserving antimicrobial functions. The reduction in inflammatory responses during endotoxin tolerance serves to preserve tissue integrity and macrophage viability, although it can also result in immunosuppression, as observed in sepsis. An enhanced understanding of the molecular mechanisms which govern endotoxin tolerance in macrophages could unlock new ways to modulate innate immunity. Here we provide detailed protocols for inducing and measuring tolerance in murine bone marrow-derived macrophages (BMDMs) and human PMA-differentiated THP-1 macrophages, using ELISA-based quantification of IL-1β and IL-6 as functional readouts. These methods provide robust in vitro systems to study endotoxin tolerance.