Receptor Interacting Protein 3-Mediated Necroptosis Promotes Lipopolysaccharide-Induced Inflammation and Acute Respiratory Distress Syndrome in Mice.

Linlin Wang,Gui Wu,Qing Liu,Zhengyuan Xia,Tingting Wang,Qingping Wu,Haobo Li,Tumenjavkhlan Socorburam,Wanli Xie,Yinglu Feng,Zhongjun Zhang

doi:10.1371/journal.pone.0155723

Abstract

Necrosis amplifies inflammation and plays important roles in acute respiratory distress syndrome (ARDS). Necroptosis is a newly identified programmed necrosis that is mediated by receptor interacting protein 3 (RIP3). However, the potential involvement and impact of necroptosis in lipopolysaccharide (LPS)-induced ARDS remains unknown. We therefore explored the role and mechanism of RIP3-mediated necroptosis in LPS-induced ARDS. Mice were instilled with increasing doses of LPS intratracheally to induce different degrees of ARDS. Lung tissues were harvested for histological and TUNEL staining and western blot for RIP3, p-RIP3, X-linked inhibitor of apoptosis protein (XIAP), mixed lineage kinase domain-like protein (MLKL), total and cleaved caspases-3/8. Then, wild-type and RIP3 knock-out mice were induced ARDS with 30 mg/kg LPS. Pulmonary cellular necrosis was labeled by the propidium Iodide (PI) staining. Levels of TNF-a, Interleukin (IL)-1β, IL-6, IL-1α, IL-10 and HMGB1, tissue myeloperoxidase (MPO) activity, neutrophil counts and total protein concentration were measured. Results showed that in high dose LPS (30mg/kg and 40mg/kg) -induced severe ARDS, RIP3 protein was increased significantly, accompanied by increases of p-RIP3 and MLKL, while in low dose LPS (10mg/kg and 20mg/kg) -induced mild ARDS, apoptosis was remarkably increased. In LPS-induced severe ARDS, RIP3 knock-out alleviated the hypothermia symptom, increased survival rate and ameliorated the lung tissue injury RIP3 depletion also attenuated LPS-induced increase in IL-1α/β, IL-6 and HMGB1 release, decreased tissue MPO activity, and reduced neutrophil influx and total protein concentration in BALF in severe ARDS. Further, RIP3 depletion reduced the necrotic cells in the lung and decreased the expression of MLKL, but had no impact on cleaved caspase-3 in LPS-induced ARDS. It is concluded that RIP3-mediated necroptosis is a major mechanism of enhanced inflammation and lung tissue injury in high dose LPS- induced severe ARDS in mice.

Highlights

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with high mortality[1]
We have shown in the current study that in high dose LPS-induced severe ARDS, lung injury was mainly due to receptor interacting protein 3 (RIP3)-mediated necroptosis, while in low dose LPS-induced mild ARDS, lung injury was mainly a consequence of caspase-dependent apoptosis
We have provided evidences that RIP3 depletion can reduce LPS-induced inflammation response, attenuate LPS-induced lung injury, and alleviate hypothermia symptoms, which eventually improve survival rate in high dose LPS-induced severe ARDS in RIP3 knock-out (RIP3-KO) mice

Summary

Introduction

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with high mortality (about 30%-50%)[1]. It is an inflammatory lung condition associated with the lung epithelium/endothelium injury and the malfunction of other organs due to the inability to take up oxygen[2]. Different types of cell death including apoptosis, autophagy and necrosis, coexist to contribute to the development of ARDS[3]. Unlike apoptosis and autophagy, which wrap intracellular contents within cells, necrosis features the release of cell lysis into the extracellular environment, and as a result, damage-associated molecular patterns (DAMPs) are released[5]. The intervention of necrosis has largely not been studied in ARDS because previous studies reported that necrosis was uncontrollable and could not be regulated

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Conclusion