The Role Of MTOR-autophagy Signaling In Asthma And PM-induced Airway Inflammation

Autophagy is a dynamic process responsible for the lysosomal degradation of damaged protein,damaged organelles,and microorganisms,whose process includes initiation,elongation,and formation of autophagosome,and autophagosome-lysosome fusion.In general,Mechanistic target of rapamycin kinase（MTOR）is known as the major negative regulator of autophagy.MTOR is a serine/threonine protein kinase belonging to the PI3K-related kinase family.It interacts with several other molecules to form two distinct complexes named MTOR complex 1（MTORC1）and MTORC2,which play distinct roles in pathophysiology.MTOR-autophagy axis plays crucial roles in major cellular processes such as metabolism and proliferation.Extensive studies have established a dominant role for MTOR in regulating cellular growth and metabolism in response to growth factors and nutrients,and reveal that MTOR signaling pathway is implicated in the progression of cancer,obesity,type 2 diabetes,as well as the aging process.Recently,we and others have demonstrated that autophagy,in some cases together with MTOR,plays pivotal roles in numerous pulmonary diseases,including chronic obstructive pulmonary disease（COPD）,acute lung injury,and pulmonary fibrosis.However,little is known about the role of MTOR-autophagy axis in asthma and PM-induced airway inflammation.Asthma is characterized by allergic airway inflammation,mucus hyperproduction,and remodeling,which eventually lead to bronchial hyperreactivity and airway obstruction.The airway epithelial cells（ECs）are recognized as an essential controller for the initiation and perpetuation of asthmatic inflammation.In response to allergens,ECs secrete endogenous danger signals such as thymic stromal lymphopoietin（TSLP）and granulocyte-macrophage colony–stimulating factor（GM-CSF）,thereby activating dendritic cells and bridging the innate and adaptive immunity.The initially recruited T cells and eosinophils secrete cytokines and chemokines and cooperatively cause further injury of ECs,which in turn produce various pro-inflammatory mediators,such as IL25,IL33,and eotaxins,to amplify and perpetuate the allergic inflammation.Thus,injury of ECs contributes significantly to asthma pathogenesis;however,the detailed mechanisms of ECs injury in asthma remain largely unknown.Air pollution is a worldwide problem affecting human health.Accumulating epidemiological and clinical studies show that exposure to air pollution,in particular airborne particulate matter（PM）,increases morbidity and mortality for respiratory diseases,such as asthma,chronic obstructive pulmonary disease（COPD）,and lung cancer.Thus,it is of great importance to explore the potential molecular mechanisms by which allergens or air pollutants trigger airway injury,in order to propose new effective therapeutic strategies.Previous study has demonstrated a genetic association of ATGs（autophagy related）with asthma and has shown increased AVs in ECs and fibroblasts from human asthmatics.CD11c-specific atg5^-/-mice displayed an IL17A-dependent neutrophilic inflammation induced by HDM.Altogether,these data suggest that the functions of autophagy in asthma pathogenesis are likely cell-specific.Moreover,several studies have indicated that inhibition of MTOR using rapamycin attenuates allergic airway inflammation,suggesting a protective role of MTOR inhibition in asthma.However,pulmonary toxicity has been widely observed when rapamycin is used as an immunosuppressive drug for renal transplantation.Thus,the roles of MTOR in asthma should also be cell type-dependent.Nevertheless,little is known about the functions of MTOR and autophagy in ECs in asthma pathogenesis.In addition,our previous study has demonstrated that autophagy is essential for environmental ultrafine PM-induced inflammation and mucus hyperproduction in airway epithelial cells.However,to the best of our knowledge,several critical issues remain unclear.What is the main function of MTOR in PM-induced damage in vitro and in vivo?Whether the function of MTOR in PM-induced airway inflammation is dependent on the downstream autophagy?Here,we aim to explore the functions of MTOR and autophagy in ECs in allergic inflammation and PM-induced airway inflammation.Part Ⅰ MTOR suppresses autophagy-mediated production of IL25 in allergic airway inflammationIntroduction Airway epithelial cells are recognized as an essential controller for the initiation and perpetuation of asthmatic inflammation,yet the detailed mechanisms remain largely unknown.This study aims to investigate the roles and mechanisms of MTOR-autophagy axis in airway epithelial injury in asthma pathogenesis.Methods We examined the MTOR-autophagy signaling in airway epithelium from asthmatic patients or allergic mice induced by ovalbumin or house dust mites,or in HBE cells.Furthermore,mice with specific MTOR knockdown in airway epithelium and autophagy-related lc3b^-/-mice were used for allergic models.Results MTOR activity was decreased,while autophagy was elevated,in airway epithelium from asthmatic patients or allergic mice,or in HBE cells treated with IL33 or IL13.These changes were associated with upstream TSC2 signaling.Specific MTOR knockdown in mouse bronchial epithelium augmented,while LC3B deletion diminished allergen-induced airway inflammation and mucus hyperproduction.The worsened inflammation caused by MTOR deficiency was also ameliorated in lc3b^-/-mice.Mechanistically,autophagy was induced later than the emergence of allergen-initiated inflammation,particularly IL33.MTOR deficiency increased,while knockout of LC3B abolished,the production of IL25 and the eventual airway inflammation upon allergen challenge.Blocking IL25 markedly attenuated the exacerbated airway inflammation in MTOR deficiency mice.Conclusion Collectively,these results demonstrate that allergen-initiated inflammation suppresses MTOR and induces autophagy in airway ECs,which results in the production of certain epithelium-derived pro-allergic cytokines such as IL25,further promoting the type 2response and eventually perpetuating airway inflammation in asthma.Part Ⅱ Inactivation of MTOR promotes autophagy-mediated epithelial injury in particulate matter-induced airway inflammationIntroduction Particulate matter（PM）is able to deposit in the respiratory tract or come into alveoli,consequently induces impairment of airway epithelial barrier,oxidative stress,autophagy,DNA damage,and genomic instability.However,the detailed molecular mechanisms mediating the adverse effects of PM remain to be further investigated.Methods We examined the MTOR-autophagy signaling in airway epithelium from mice,primary mouse tracheal epithelial cells or in HBE cells.Moreover,mice with specific MTOR or ATG5 knockdown in airway epithelium were used for PM-induced model in vivo,while HBE cells were treated with si RNA or inhibitors to inactivate the MTOR-autophagy signaling in vitro.Results PM exposure inactivated MTOR,enhanced macroautophagy/autophagy,and impaired lysosomal activity in HBE cells and in mouse airway epithelium.Genetic or pharmaceutical inhibition of MTOR significantly enhanced,while inhibition of autophagy attenuated,PM-induced IL6 expression in HBE cells.Consistently,club-cellspecific deletion of Mtor aggravated,whereas loss of Atg5 in bronchial epithelium reduced,PM-induced airway inflammation.Interestingly,the augmented inflammatory responses caused by MTOR deficiency were markedly attenuated by blockage of downstream autophagy both in vitro and in vivo.Mechanistically,the dysregulation of MTOR-autophagy signaling was partially dependent on activation of upstream TSC2,and interacted with the TLR4-MYD88 to orchestrate the downstream NFKB activity and to regulate the production of inflammatory cytokines in airway epithelium.Moreover,inhibition of autophagy reduced the expression of EPS15 and the subsequent endocytosis of PM.Conclusion Taken together,the present study provides a mechanistic explanation for how airway epithelium localized MTOR-autophagy axis regulates PM-induced airway injury,suggesting that activation of MTOR and/or suppression of autophagy in local airway might be effective therapeutic strategies for PM-related airway disorders.