Therapeutic efficacies of neutrophil elastase and RIP1 inhibitors in acute lung injury

5. 项目总结/文摘

5. Project Summary/Abstract Acute lung injury (ALI) is a syndrome of respiratory failure characterized by protein-rich pulmonary edema that causes severe hypoxemia and labored breathing. In the United States, the annual incidence of ALI is about 200,000 with a mortality rate of 30-40%; yet, there is no FDA-approved pharmacological therapy for ALI. Neutrophils are the most abundant circulating white blood cells in the human body and provide the first line of defense against invading pathogens. In the lung, while neutrophils are essential for pathogen elimination, exuberant accumulation and prolonged survival of neutrophils in the interstitial and alveolar spaces contribute to ALI by releasing a variety of injurious molecules, including neutrophil elastase (NE), metalloproteases, reactive oxygen species (ROS), and other proteolytic enzymes. Although all these injurious molecules contribute to ALI, an increasing body of studies have demonstrated that NE is the most potent hydrolytic enzyme that plays a key role in lung alveolar injury, and that inhibition of NE with small molecular inhibitors [e.g. Sivelestat (Siv)] reduces measures of lung injury and inflammation in animal models of ALI. However, clinical studies have not yet provided a clear consensus. Whereas in Japanese phase III clinical trials, Siv improved pulmonary functions, in North American multicenter studies, Siv rather increased long-term mortality by unknown mechanisms. Thus, identification of molecular events associated with adverse effects of Siv in ALI is an important task for the development of novel and combination treatment regimens. Recently, in unbiased functional protease purification experiments, we discovered that NE cleaves receptor interacting protein 1 (RIP1), and that inhibition of NE with Siv leads to RIP1-dependent necroptosis in mouse and human neutrophils following treatment with lipopolysaccharide (LPS) or TNFα. Such RIP1-induced necroptosis can be inhibited by RIP1 kinase inhibitors (e.g. Nec-1s) or knockout (KO) of RIP3. We found that LPS/Siv-induced necroptosis was significantly inhibited in RIP3 KO neutrophils. Importantly, in a mouse model of ALI induced by 50% lethal dose of LPS, Siv in combination with Nec-1s resulted in 90% survival of the mice (P=0.001), whereas neither Siv nor Nec-1s alone afforded significant protection against the death. Thus, we hypothesize that inhibition of NE with Siv in ALI leads to RIP1-mediated necroptosis in neutrophils, which causes secondary inflammation and lung injury, and that inhibition of RIP1-mediated necroptosis significantly improves the effectiveness of NE inhibitors in ALI. Aim-1. Determine the therapeutic efficacies of RIP1 and NE inhibitors in lung injury and inflammation using mouse models of ALI. Aim-2. Determine how genetic ablation of NE or RIP3 affects lung injury and inflammation in ALI mice.