Abstract P131: Na/k-atpase Is A Negative Regulator Of Lps-induced Macrophage Activation

Abstract

Introduction and objective: A strong association between atherosclerosis and infections has been suggested in several studies. Clarifying the mechanism of macrophage inflammatory activation is critical to reduce the risk of atherosclerotic cardiovascular disease. Recent studies indicate that the Na/K-ATPase (NKA) is a novel regulator of macrophage activation. Nevertheless, its role in bacterial lipopolysaccharide (LPS)-induced innate immune responses remains unclear. In this study, we aim to investigate how NKA regulates LPS signaling and macrophage inflammatory activation. Methods and Results: Using murine peritoneal macrophages isolated from genetically modified mice, we showed that macrophages partially deficient in NKA α1 were hypersensitive to LPS. 100 ng/ml LPS triggered enhanced pro-inflammatory cytokine production such as IL-1β, IL-6, MCP-1 and TNF-α through RT-PCR and Elisa assay. Furthermore, intraperitoneal injection of LPS inducing septic shock in mice resulted in higher plasma pro-inflammatory cytokine levels and lower survival rate in NKA α1 heterozygous null mice. Mechanistically, co-IP experiments showed that TLR4 assembles with NKA and phosphorylated-Lyn as a complex in response to LPS in WT macrophages and reduction in NKA α1 led to more co-precipitated p-Lyn by LPS stimulation. Moreover, the Src-family kinases inhibitor PP2 blocked the production of pro-inflammatory cytokine from post culture medium of LPS treated WT macrophages. In addition, RNA sequencing and Gene Set Enrichment Analysis showed an upregulation of NF-κB target genes. The activation and nuclear translocation of NF-κB were also augmented. Conclusions: NKA is a novel negative regulator of LPS-mediated inflammatory activation by regulating NKA α1/TLR4/Lyn Complex and NF-κB signaling pathway in macrophages.