An Original Model Based On TCRPs For NETosis Analysis And Its Functional Assessment On Lung Epithelial Injury

NETosis,the dynamic process to release Neutrophil extracellular traps(NETs),is a new-found antibacterial way of activated neutrophils during inflammation.When stimulated by microorganisms or other factors,the spherical neutrophils become flat,and nuclear membrane and cell membrane rupture,then nucleic acid materials such as DNA fuse with cytoplasmic granulosa protein.The mixture are released into extracellular environment,forming NETs.NETs contain high concentrations of proteolytic enzymes that could trap and kill bacteria.However,excessive formation of NETs inevitably damage host tissue.Acute lung injury(ALI),with high incidence and mortality,is the destroy of alveolar epithelial cells and capillary endothelial cells due to various detrimental factors stimulation,which leads to acute hypoxia and respiratory insufficiency.It has been reported that the pathogenesis of ALI is related to the intemperate activation and accumulation neutrophils in the lung tissue,and the concentration of free DNA in peripheral blood of ALI patients was higher than healthy persons.However,whether the toxicity of activated neutrophils is ascribed to NETs release and whether reducing NETs generation can relieve tissue injury were remained to be clarified.In addition,there is still a lack of clinical drugs targeted for ALI treatment.Real-time cell analysis(RTCA)systems based on electrical micro impedance can monitor the time/dose-dependent cell response patterns(TCRPs)of targeted cells under various stimuli.In this study,using xCELLigence laboratory apparatus,we applied TCRPs technology to established a new method for NETosis dynamic evaluation:Firstly,we established a stable lung epithelial barrier in RTCA system by optimizing cell density according to the growth curve differences.Neutrophils under various treatments were then added into the lung epithelial barrier at platform stage,and the micro-impedance signal of lung epithelial cells was continuously recorded by being co-cultured with neutrophils.The status of neutrophils were dynamically indirectly reflected by CI change.The TCRPs results showed that LPS-activated neutrophils destroyed lung epithelial cell cytoskeleton,and caused cell apoptosis,thus inducing epithelial barrier damage and Cell Index(CI)curve decline.Subsequently,similar CI decline and cell damage effects were observed when extracted NETs were added into the lung epithelial barrier at plateform stage.To further explore whether the cytotoxity of activated neutrophils is due to NETs release,we pre-treated neutrophils with NADPH inhibitor DPI to block NETosis process.As the results showed,the lung epithelial damage effects and Cl decline was significantly alleviated by co-cultured with DPI-pretreated neutrophils,which were different from the severe loss caused by LPS-activated neutrophils.Therefore,it was concluded that NETs release was one of the main reasons for activated neutrophils cytotoxity.By monitoring lung epithelial barrier Cl dynamical changes,the TCRPs-based analysis technology could reflect the NETosis induction/reduction in real-time.Our established NETosis dynamical analysis method is able to evaluate the epithelial damage difference caused by neutrophils under different conditions or from various sources,so as to assess the difference in NETs release levels.Next,we want to further explore whether this established detection method has clinical application value in the diagnosis and treatment of ALI diseases.As is well known,the main cause of ALI is pulmonary inflammation and infection,so we focused on patients with pneumonia as research subjects.By comparing clinical venous blood samples,we found that neutrophils from severe pneumonia patients peripheral blood induced more severe lung epithelium CI decline comparing to healthy controls,which was related to their difference in NETs formation level,as identified by other common NETs detection methods.Since NETs was formed in inflammatory environment after neutrophils stimulated by inflammatory stimuli,so we try to detect glucocorticoid and several common non-steroidal anti-inflammatory drugs on NETosis effect.The result showed that aspirin(ASA)and acetaminophen(APAP)could inhibit NETs release to some extent,thus easing lung epithelial injury.The conclusion was consistent with others’ research results,which further proved the feasibility of microelectronics RTCA application into NETosis measurement.Meanwhile,it indicated this method had potential clinical application value.In short,we created a real-time NETosis analysis model based on TCRPs technology,which possessed the following advantages:no labels,dynamics and continuity,high-throughput,comparing with traditional method.This quantitative evaluation for activated neutrophils or released NETs damage on host tissue contributes to the further explain of ALI pathogenesis,and facilitates subsequently wide screening for NETs inhibiting drugs,promoting the treatment of ALI.