Functions And Mechanisms Of Targeting Eosinophil Ferroptosis In Alleviating Asthmatic Airway Inflammation

Background:Asthma is a chronic airway inflammation disease in which many kinds of cells and cellular elements are involved,and it is one of the most common chronic respiratory diseases in the world.As a specific type of immune cells in asthmatic airway inflammation,eosinophils（Eos）are considered to be one of the most critical effector cells for the pathogenesis of asthma.In the normal status,circulating Eos usually end physiologically by spontaneous apoptosis within 2-5 days;while in the asthmatic inflammatory foci,the lifespan of Eos is believed to be prolonged due to the presence of pro-survival factors in the local inflammatory microenvironment.An increasing number of studies have proposed that delaying apoptosis of Eos is a significant mechanism for its accumulation at the inflammatory site,which will impede the resolution of allergic inflammation and delay the restoration of tissue homeostasis.Thus,selective induction of Eos death is likely to be effective for controlling the asthmatic inflammation.Moreover,our group has firstly discovered that the inhibitors of anti-apoptosis protein Bcl-2 could reduce allergic inflammation by promoting Eos apoptosis（J Allergy Clin Immunol,2017）.Ferroptosis,a novel form of nonapoptotic cell death,is characterized by the accumulation of iron-dependent lipid peroxidation.Overwhelming evidence have revealed the close relationship between ferroptosis and the pathological process of several clinical diseases.However,the relationship between ferroptosis and asthma is still not clear at present.A recent study has suggested that Eos have a higher level of iron storage than other inflammatory cells.Combined with the previous reports that cellular iron overload easily leads to the accumulation of lipid peroxidation,which results in cell ferroptosis eventually.All these studies indicate that Eos seem to be more sensitive to the ferroptosis stimulation due to its high level of iron.Thus,in this study,we will investigate the potential relationship between ferroptosis and Eos,and further explore the application value of ferroptosis in the management of asthma.Objective:To investigate whether treatment of ferroptosis-inducing agents could trigger Eos ferroptosis and eventually relieve the established asthmatic airway inflammation,and to further clarify their potential synergistic effects with glucocorticoids.Methods:To explore whether the stimulation of ferroptosis-inducing agents could lead to Eos death in vitro,we initially harvested human Eos from peripheral blood of asthmatic or non-asthmatic patients,and mouse Eos from peripheral blood and bronchoalveolar lavage fluid（BALF）.Human and mouse Eos were then incubated with a series of ferroptosis-inducing agents and cell viability was assessed by flow cytometry.Next,to determine the specific manner of Eos death caused by ferroptosis-inducing agents,we used transmission electron microscopy to directly examine the morphological changes after cell death.And small-molecule inhibitors of apoptosis,necrosis,autophagy or ferroptosis were further used to examine their effects on cell death triggered by ferroptosis-inducing agents.Moreover,we stained Eos with C11-BODIPY,CM-H₂DCFDA,and Mito SOX to detect the changes of lipid ROS,cytosolic ROS,and mitochondrial ROS respectively in Eos stimulated with ferroptosis-inducing agents.In order to verify the type of lethal ROS that mediating Eos death,we further applied the specific antioxidants to examine the functional requirement of different types of ROS in ferroptosis-inducing agents triggered cell death.We finally explored whether facilitating Eos ferroptosis would attenuate allergic airway inflammation in vivo.We established the classical animal model of asthma and administrated with ferroptosis-inducing agents by intraperitoneal injection.The BALF and lung tissues were collected for examining the airway inflammation and BALF Eos viability by cell counting,flow cytometry,and quantitative real-time PCR.In addition,we further co-administrated ferroptosis-inducing agents with dexamethasone（DXMS）in vitro and in vivo to investigate the combination efficacy of ferroptosis-inducing agents and glucocorticoids in Eos death and mouse models of allergic airway inflammation.Results:In this study,we demonstrated ferroptosis-inducing agents could obviously induce cell death of Eos in a time-and concentration-dependent manner in vitro.Further investigation on the manner of cell death revealed that Eos ferroptosis triggered by ferroptosis-inducing agents occurred in a non-canonical pathway:on the one hand,the morphological results indicated that the typical morphological changes of cell ferroptosis were observed in ferroptosis-inducing agents-treated Eos.Additionally,we found that Eos death triggered by ferroptosis-inducing agents was iron-dependent.On the other hand,unlike the canonical pathway of ferroptosis,we have found here that lipid ROS accumulation induced by ferroptosis-inducing agents was not the lethal factor of Eos ferroptosis,and further analysis verified that the increase of cytosolic ROS was the principal cause of cell death.In the established mouse model of asthma,we demonstrated that promoting the ferroptosis of Eos effectively attenuated eosinophilic airway inflammation of asthma.More importantly,ferroptosis-inducing agents exerted a synergistic efficacy when combined with DXMS on Eos death in vitro,and the combined administration could also improve the therapeutic effect and reduce the dosage of DXMS in vivo.Conclusion:Ferroptosis-inducing agents could effectively relieve asthmatic airway inflammation via promoting non-canonical ferroptosis of Eos,suggesting new targets and ideas for the development of medicines for asthma therapy.In addition,the synergistic efficacy when combining ferroptosis-inducing agents with glucocorticoids further provides a new therapeutic target for the treatment of refractory asthma patients with steroid tolerance or resistance.