Necroptosis in pulmonary macrophages mediates lipopolysaccharide-induced lung inflammatory injury by activating ZBP-1

Abstract

Z-DNA combined protein-1 (ZBP-1), an important necroptosis regulator, activates necrosis-associated inflammation and immune response. Increased ZBP-1 expression in necroptosis-associated inflammation correlates with activation of receptor interacting protein kinase (RIPK1)/RIPK3 and nuclear factor (NF)-κB. Here we explored the role of ZBP-1-mediated necroptosis in lipopolysaccharide (LPS)-induced lung injury. Bone marrow-derived macrophages (BMDMs) transfected with a small interfering RNA against ZBP-1 or scrambled control RNA were administered to mice that had been depleted of alveolar macrophages (AMs). Then the animals were treated with E. coli LPS (2.0 mg/kg) or phosphate-buffered saline by intratracheal instillation for 48 h. LPS-induced lung inflammatory injury was verified, and the mRNA and protein expression of ZBP-1, RIPK1/RIPK3 and NF-κB in AMs were then assessed by Western blot and real time-quantitative polymerase chain reaction. In mechanistic studies in vitro, BMDM cultures were treated with different concentrations of LPS for 24 h, and the expression of ZBP-1, RIPK1/RIPK3 and NF-κB were assessed. LPS activated ZBP-1-mediated necroptosis, primarily in AMs. This activation and associated lung inflammatory injury were much weaker after AMs depletion or silencing of ZBP-1 in BMDMs, which correlated with down-regulation of RIPK1/RIPK3. These in vivo findings were confirmed in experiments with cultures of BMDMs. In conclusion, LPS induces lung inflammation and injury by activating ZBP-1-mediated necroptosis and release of pro-inflammatory cytokines by macrophages.