The NLRP3 inflammasome in macrophages is stimulated by cell-free hemoglobin

Cell-free hemoglobin (CFH) is associated with severe lung injury in human patients and is sufficient to induce airspace inflammation and alveolar-capillary barrier dysfunction in an experimental model of acute lung injury. The mechanisms through which this occurs are unknown. One key pathway which regulates inflammation during acute lung injury is the NLRP3 inflammasome. Because CFH can act as a damage-associated molecular pattern, we hypothesized that CFH may activate the NLRP3 inflammasome during acute lung injury. Primary mouse alveolar macrophages and cultured murine macrophages exposed to CFH (0-1 mg/ml) for 24 hr demonstrated robust upregulation of the NLRP3 inflammasome components NLRP3, caspase-1, and caspase-11. Maximal induction of the NLRP3 inflammasome by CFH required TLR4. Compared to wild-type controls, mice lacking NLRP3 developed less airspace inflammation (2.7 × 10⁵  cells/ml in bronchoalveolar lavage fluid versus. 1.1 × 10⁵ /ml, p = .006) after exposure to intratracheal CFH. Together, these data demonstrate that CFH can stimulate the NLRP3 inflammasome in macrophages and that this pathway may be important in the pathogenesis of CFH-induced acute lung injury.