Inducing Ferroptosis-Like Cell Death Of Neutrophils Alleviates Airway Inflammation

Background: asthma is a chronic airway inflammatory disease,which involves in many kinds of cells(eosinophils,neutrophils,mast cells,T cells,etc.)and cell components.Although infitrative function of eosinophils and mast cells in the occurrence and development of the disease has been recognized,more and more studies have also evaluated the infiltration of neutrophils.Patients with increasing number of neutrophils in the airway are resistant to the treatment of corticosteroids.Therefore,it is important to explore underlying pathways which unlike traditional treatment strategy.Acute lung injury(ALI)or acute respiratory distress syndrome(ARDS)refers to the disseminated pulmonary interstitial and alveolar edema which caused by the injury of pulmonary capillary endothelial cells and alveolar epithelial cells in the process of various non-cardiogenic diseases,resulting in hypoxic respiratory insufficiency or failure.Infection of pathogen leads to the normal lung-blood barrier disfunction and diffuse pulmonary interstitial and alveolar edema,accompanied by a large number of leukocytes,especially neutrophils infiltration.Induction death of neutrophil is of great significance to attenuate the symptoms of ALI,control the further development of ALI and reduce the mortality.Ferroptosis is an iron-dependent death,which caused by accumulation of lipid peroxidation in cells.It is different from other death modes in morphology,biochemistry,heredity and so on.We believe that the inducing neutrophils ferroptosis may be a potential way to alleviate airway neutrophil inflammation.Objective: To investigate whether RSL3 can induce neutrophil ferroptosis and protect airway inflammation.Methods: Neutrophils were collected from peripheral blood of patients with acute lung injury or asthma and bronchoalveolar lavage fluid(BALF)of ALI model mice,and the effects of RSL3 on neutrophil survival were observed.The effects of ferroptosis inhibitors(DFO and ferrostatin-1),autophagy inhibitor(Spautin-1),apoptosis inhibitor(Z-VAD-FMK),necroptosis inhibitor(Necrotatin),total antioxidants(NAC,GSH)in combination of RSL3 were examined.The changes of cell morphology were observed under electron microscope,and examined lipid reactive oxygen species(ROS),mitochondrial ROS and cytoplasmic ROS.C57 mice were randomly divided into control group and RSL3 group.Ovalbumin Grade V/ Complete Freund's adjuvant(OVA/CFA)model and Lipopolysaccharide(LPS)model were established.RSL3 was injected intraperitoneally and the number of BALF total cells and neutrophils,the expression of inflammatory cytokines in lung tissue and the infiltration of lung inflammation were detected.Results: RSL3 induced ferroptosis-like death in neutrophils,which was time-and dose-dependent.Ferroptosis inhibitor(DFO,ferrostatin-1),autophagy inhibitor(Spautin-1)and comprehensive antioxidant(NAC,GSH)could alleviate cell death induced by RSL3.ROS level increased gradually after RSL3 intervention,and mitochondrial ROS participated in the more important part.After intraperitoneal injection of RSL3 in OVA/ CFA model and LPS model,the total number of BALF cells,the proportion and number of neutrophils,the level of IL-6 and cxcl-2 inflammatory factor m RNA in lung tissue,and the inflammatory infiltration in lung tissue were decreased.Conclusion: RSL3 can induce neutrophil ferroptosis,autophagy and mitochondrial ROS are involved in RSL3 induced cell death.Induction neutrophil ferroptosis can alleviate neutrophil airway inflammation and provide reference for clinical treatment of steroid resistant asthma and acute lung injury.