Mechanism Of Alveolar Epithelial-derived Exosomes Promoted M2 Macrophage Polarization In The Early Stage Of Mechanical Ventilation

Objective: Mechanical ventilation is a necessary means of respiratory support,and ventilator-induced lung injury is the most common complication,which seriously affects the prognosis and living quality of patients.This study aims to explore the effect of alveolar epithelial cell-derived exosomes on the M2 polarization of macrophages in the early stage of mechanical ventilation by in vivo and in vitro and clarify the molecular mechanism that alveolar epithelial cell-derived exosomal miR-21a-5p regulates M2 macrophage polarization.Methods:(1)Mouse model of mechanical ventilation with high and low tidal volume ventilation was established,and the concentration of inflammatory factors and macrophage polarization in bronchoalveolar lavage fluid were detected.(2)The exosomes were extracted from the non-cyclical-stretched/cyclical-stretched alveolar epithelial cells and indentified.The exosomes were added into macrophages to observe the uptake of exosomes by macrophages and detected the polarization of macrophages.(3)The expression level of miR-21a-5p in exosomes was detected.miR-21a-5p was inhibited before adding exosomes,and macrophage proportion were detected.Target gene Notch2 of miR-21a-5p was predicted by Target Scan website and verified by dual luciferase reporter gene and transfection of miR-21a-5p mimic.miR-21a-5p was inhibited before adding exosomes,and the expression of Notch2 was detected.Notch2 ligand Jagged-1 was used to activate Notch2 to detect macrophage polarization.The differentially expressed genes related to macrophage polarization in macrophages that treated with exosomes derived from non-stretched / stretched alveolar epithelial cells were screened by RNA-Seq,and the interaction between Notch2 and differentially expressed genes was verified by CO-IP.Knockdown SOCS1 by siRNA and followed by Notch2 activation using Jagged-1 to verify that Notch2 / SOCS1 was involved in the regulation of M2 macrophage polarization.(4)The exosomes derived from non-cyclical-stretched/ cyclicalstretched alveolar epithelial cells were injected into the lung tissue of mice,and the expression of miR-21a-5p and Notch2 were detected,and the polarization of macrophage was also detected.miR-21a-5p antagomir was instilled into the lung tissue of mice.The expression of Notch2/SOCS1 and the M2 macrophages proportion in bronchoalveolar lavage fluid were detected.Mice were treated with miR-21a-5p antagomir before ventilation,and the M2 macrophages proportion in bronchoalveolar lavage fluid was detected.The exosome miR-21a-5p derived from alveolar epithelial cells at the early stage of mechanical ventilation promoted the polarization of M2 macrophages through Notch2 /SOCS1 in vivo.Results:(1)High / low tidal volume ventilation did not cause significant inflammatory changes in the lungs tissue of mice,but increased the polarization of M2 macrophages in alveolar lavage fluid.(2)Cyclic stretching induced alveolar epithelial cells releasing exosomes which were taken up by macrophages and promoted M2 polarization of macrophage and decreased M1 polarization.(3)The expression of miR-21a-5p in exosomes derived from stretched alveolar epithelial cells was significantly increased,and the expression of miR-21a-5p in macrophages was also increased after treated with exosomes.miR-21a-5p inhibition reduced M2 macrophages polarization.The results of dual-luciferase reporter gene showed that wild-type Notch2 reduced the fluorescence activity,while mutant Notch2 restored the fluorescence activity,and transfection of miR-21a-5p mimic also reduced the expression of Notch2.miR-21a-5p inhibition increased the expression of Notch2.Activation of Notch2 by Jagged-1 reduced M2 macrophage polarization.RNA-Seq screened out the differentially expressed gene SOCS1 which was correlated with macrophage polarization in macrophages after treated with exosomes,and confirmed the interaction between Notch2 and SOCS1 by CO-IP.The exosomes-derived from cyclically stretched epithelial cells decreased SOCS1 protein expression in macrophages.Knockdown of SOCS1 with siRNA followed by activation of Notch2 with Jagged-1 increased M2 macrophage polarization.(4)Exosomes-derived from stretchedalveolar epithelial increased the M2 macrophages proportion in bronchoalveolar lavage fluid of mice.The expression of miR-21a-5p and the surface markers of M2 macrophages were also increased.The expression of surface markers of M1 macrophages and the protein expression of Notch2 were decreased in lung tissues of mice.The proportion of M2 macrophages was reduced after instillation of miR-21a-5p inhibitor antagomir;the proportion of M2 macrophages was also decreased after administrated with miR-21a-5p antagomir before mechanical ventilation.Conclusion: This study found that cyclically stretched alveolar epithelial cells could release exosomes which encapsulated miR-21a-5p and delivered to macrophages,and promoted M2 macrophage polarization by inhibiting Notch2/SOCS1.Experiments in vivo confirmed that the lungs were still in a steady-state in the early period of mechanical ventilation,and exosomes coud be transfered from alveolar epithelial cells to macrophages,promoted macrophages in the anti-inflammatory phenotype M2 which maintain lung homeostasis.The results of this experiment indicated that exosomes could be served as a novel strategy for the prevention and treatment of lung injury induced by mechanical ventilation,and Notch2/SOCS1 could be served as a therapeutic target for lung injury induced by mechanical ventilation.