Study On The Role Of ILCs In The Mechanism Of Airway Inflammation Induced By DEPs In Mice

Background and purposeWith the development of China’s economy and urbanization,outdoor air pollution has become a serious public health problem in large cities of China,especially related to the incidence of respiratory diseases.Among the outdoor air pollutants in China,the fine particulate matter(PM2.5)exceeds the standard most obviously.And motor vehicle exhaust is an extremely important part of the source of PM2.5 in the city,Therefore,it has great practical significance to clarify the mechanism of action of PM2.5 on the health effects of respiratory system.The source and composition of vehicle exhaust particulates are very complicated,and this mainly comes from diesel exhaust particles(DEPs);As the report of "2017 China Motor Vehicle Pollution Prevention and Control Annual Report" posted by China’s Ministry of Environmental Protection,DEPs contributes more than 90%of the total vehicle emissions.Therefore,the PM2.5 source of motor vehicles in the outdoor atmosphere is mainly dominated by Diesel exhaust particles(DEPs).In recent years,a large number of epidemiological investigations at home and abroad have confirmed that motor vehicle exhaust can induce a variety of airway inflammation,which are closely related to the increase of cough,bronchial asthma attack and lung function index.In the study of cell and animal models,the effects of DEPs on airway inflammation have been initially confirmed.DEPs can induce inflammatory response and oxidative stress in human airway epithelial cells;however,DEPs can produce different type inflammation under different animal experimental conditions,such as Granulocyte inflammation,Eosinophilic inflammation,or even the emergence of airway hyperresponsiveness(AHR)and other results.Therefore,this study will first explore what type of airway inflammation will occur in diesel exhaust particulates and whether there will be airway hyperresponsiveness.Innate Lymphoid Cells(ILCs)are a new class of immune cell subsets newly discovered in recent years.ILCs still belong to the lymphocyte lineage in development and morphology,but they do not express antigenspecific recognition receptors(TCR or BCR),which can be divided into three categories:type Ⅰ ILC(ILC1),type Ⅱ ILC(ILC2),and Type Ⅲ ILC(ILC3),in which ILC2 mainly mediates Th2-type inflammatory response,and ILC3 mainly mediates Th17 type inflammatory response.Recent studies have revealed that ILC2 and ILC3 can be independent of adaptive immunity and play an important role in regulating airway inflammation and AHR.However,it is rarely reported which type of airway inflammation is produced in mice and whether AHR produced or ILC2/ILC3 play a role under exposure of diesel exhaust which simulated the real-world conditions.Our study will attempt to construct a diesel exhaust chamber that simulates the real environment,explore the type of airway inflammation produced in mice and study the mechanism of action of ILC2/ILC3.Research method1.Establish a diesel exhaust exposure laboratory,and extract the analytical components of DEPs and prepare them for lyophilized powder for storage.2.Inject the DEPs solution by tracheal intubation(3 times/week,3 weeks,6 mg/kg),and set up a blank control group and a solvent group to detect the degree and type of airway inflammation and detect whether there is AHR occurred.3.Direct exposure in the diesel engine exposure laboratory,set different exposure time(2 weeks,4 weeks,6 weeks)and different concentrations(high:500ug/m3;Iow:200ug/m3)to detect the difference in airway and lung inflammation degree and type and airway responsiveness.The expression of ILC2/3 was detected and its correlation was analyzed.4.Based on the conditions of the best modeling in the previous step,the ILCs inhibitor group and the CD4+T cell inhibitor group were established to explore the role of ILC2/3 in the inflammatory response of the animal.5.The sensitized mice were placed in the diesel exhaust laboratory for short-term exposure to construct an acute asthma model and detect the expression of IL-17+ILCs and IL-17+CD4+T.At the same time,a blank control group,ILCs inhibitor group and CD4+T cell inhibitor group were established for studied on the role of IL-17+ILCs in this animal model.Research result1.The collected DEPs were formulated into a 3 mg/ml solution and administered to the tracheal intubation(3 times/week,3 weeks,6 mg/kg)for 3 weeks.The results showed that the mice only produced neutrophil infiltration.Airway inflammation,no AHR production.2.In the laboratory environment of diesel tail exposure,the BALB/C mice under the high concentration of 4 or 6 weeks exposure showed a significant increase in submucosal eosinophil infiltration in its peripheral airway,while the low concentration and short duration,the mice showed no obvious eosinophil infiltration.The expression of IL-5 and IL-17A in lung tissue was significantly increased,and in 4 weeks.The presence of AHR was shown in the high concentration of the exposed group;the IL-5+ILC2 and IL-17A+ILCs ratio was significantly higher than that ofCD4+T cells,and the expression was highest in the 4-week group.3.In the BALB/C mice exposed to diesel exhaust for 4 weeks,the airway inflammation infiltration in the ILCs-suppressed group was significantly reduced,and no obvious eosinophils were observed.The airway resistance test was significantly lower than that in the positive control group.In the CD4+T cell suppression group,the airway and pulmonary inflammatory infiltration were slightly attenuated compared with the positive control group in the absence of CD4+T cells,and a small amount of eosinophil infiltration was still observed in the peripheral airway mucosa.Airway resistance was not reduced compared with the positive control group.In the absence of CD4+T cells,ILC secreted IL-5 and IL-17A significantly increased.4.After sensitized mice in the environment of diesel exhaust exposure chamber,eosinophilic and neutrophil infiltration can be seen in airway and lung tissue,airway responsiveness is significantly increased,lung tissue IL-5,IL-17A of lung tissue was significantly elevated.Flow cytometry showed that IL-17+ILCs and IL-17+CD4+T in lung tissue of mice were significantly higher than those in the control group,and IL-17+ILCs expression was significantly higher than IL-17+CD4+T.Pulmonary inflammation was significantly reduced in the ILCs inhibitor group,and there was no difference in AHR compared with the blank group.Conclusion1.Instillation of mice with the collected particulate solution alone can lead to non-specific airway inflammation,but does not produce eosinophilic infiltration and AHR.2.Under certain circumstances of the chamber of diesel exhaust,mice can produce Th2/Th17 mixed airway inflammation under non-sensitized conditions,accompanied by AHR production;ILCs play a more important role in the process of inflammation and AHR production than CD4+T cells,and ILCs can mediate airway inflammation in mice induced by DEP exposure independent of CD4+T cells.3.In the early stage of asthmatic induction of sensitized mice with diesel exhaust,the secretion of IL-17 was mainly derived from ILCs rather than Th17 cells,suggesting that ILCs play a more important role in this type of asthma.