The Protective Effect And Mechanism Of Targetedly Inhibition Of Bronchial Epithelial Cell CGAS Expression On Bronchial Asthma

Bronchial asthma?also referred as asthma?,is one of the most common chronic airway inflammatory diseases worldwide,which affects over 300 million people globally and over 45.7 million people in China,which increases huge medical burden.Asthma is a heterogenous disease,which requires personalized treatment approaches based on the specific clinial situations of patients.With a variety of theories,the molecular mechanism of asthma has not been fully illustrated yet due to its complexity and heterogeneity.Recently,the role of airway epithelial cells?ECs?in asthma pathogenesis gradually arose great attentions.Previous studies identified apoptosis of ECs are likely to be induced when airways of asthmatic patients being exposed to triggers like atmospheric pollutants,allergens and pathogens.Further studies figured out that the leaking apoptotic DNA can trigger a series of immuno-inflammatory responses.DNA released into cytoplasm would be recognized by cytosolic DNA sensor and therefore activates innate immune signaling pathways.The innate immunity defense system is then activated by the translation and expression of type I IFN and pro-inflammatory cytokines,along with the enhancement of acquired immune response.Cyclic guanosine monophosphate?GMP?–adenosine monophosphate?AMP?synthase?cGAS?is a newly discovered DNA sensor that detects cytosolic DNA as a DAMP.The range of double strand DNA?dsDNA?includes exogenous virus DNA,plasmid DNA,apop DNA.Here we hypothesized that cGAS may play a key role through sensing cytosolic DNA of airway epithelium and subsequently triggering Th2-immune response in asthma pathogenesis.Objective:Our study aims to investigate the role of bronchial epithelial cGAS plays in bronchial asthma pathogenesis and further explore the specific mechanisms.Methods:Part 1The cytosolic dsDNA accumulation in epithelial cells of OVA-induced short time asthmatic model in vivo and IL-33-treated HBEs in vitro were measured by immunofluorescence.Besides,CC10-rt TAtg/-or?tet O?7CMV-Cretg/-transgenic mice were mated with cGAS^flox/flox mice to generate double-transgenic CC10-rt TA/cGASflox/flox or?tet O?7-Cre/cGAS^flox/flox mice.Then,these 2 types of mice were then mated to generate CC10-rt TA/?tet O?7-Cre/cGAS^flox/flox triple-transgenic mice(also referred as cGAS^?/?mice).6-8weeks old cGAS^?/?mice and littermate control group mice were used for OVA traditional asthma model.Then we measured the total number of inflammatory cells,the number and percentage of eosinophils in bronchoalveolar lavage fluid?BALF?,airway inflammation infiltration level,AHR and mucus secretion level to identify the role of cGAS played in asthma phenotype.Part 2The m RNA and protein level of inflammatory cytokines in OVA-induced short time point model and classical model were tested by RT-q PCR and ELISA.HBEs were stimulated by IL-33 in vitro to simulate asthmatic epithelial cells.The effects of cGAS knockout on IL-33 induced GM-CSF production.Mitochondrial specific antioxidants Mito-TEMPO was used to intervene the IL-33-treated HBEs to explore the cytosolic dsDNA abundance and GM-CSF production.Results:Part 1Increased accumulation of cytosolic dsDNA in the early stage was observed in both airway epithelium cells of OVA-challenged mice and IL-33-treated HBEs.The IL-33-induced dsDNA were colocalized with cGAS.Deletion of cGAS in the airway ECs of mice significantly attenuated the allergic airway inflammation induced by OVA,including BALF total number of inflammatory cells,the number and percentage of eosinophils,AHR and mucus secretion level.Part 2The expression level of IL-4,IL-5,IL-13,IL-25,IL-33 in cGAS^?/?group were significantly decreased in classical OVA asthmatic model.While m RNA level of GM-CSF instead of IL-25 or IL-33 significantly decreased in OVA short time point model.The m RNA expression level of GM-CSF in HBEs was significantly induced by IL-33 in vitro.IL-33-induced GM-CSF production was also significantly alleviated by cGAS knockout on HBEs.Mito-TEMPO was found to significantly reduce the IL-33-induced cytoplasmic dsDNA accumulation and GM-CSF m RNA level elevation in HBEs.Conclusion:In conclusion,airway epithelial cGAS plays an important role in asthma pathogenesis.Mechanically,cGAS participates in promoting Th2 immunity likely via sensing the cytosolic mitochondial DNA and further regulating the production of airway epithelial GM-CSF.