Role Of IRAK-M In The Biological Behavior Changes Of Airway Epithelial And Fibroblast Cells Induced By Cigarette Smoke Extrac

Part1:Role of IRAK-M in the development of airway epithelial inflammation and the potential molecular mechanism investigationBackground and Objective:Interleukin-1 receptor associated kinase-M(IRAK-M),a negative regulator of TLR pathway,has been shown to play anti-/pro-inflammatory roles in various diseases.Previous studies on IRAK-M mainly focused on innate immune cells such as macrophages and dendritic cells.However,IRAK-M is also abundantly expressed in airway epithelial cells.We have previously reported that IRAK-M can suppress airway inflammation through regulation of immune cells,such as T cell subsets,dendritic cells,and macrophages,in mice under acute cigarette smoke and LPS exposure.Nevertheless,the direct impact of IRAK-M on epithelial cells remains unclear.Given that smoking is the most important risk factor for COPD,our study aims to investigate the direct regulatory effect and potential mechanisms of IRAK-M on cigarette smoke-induced airway epithelial inflammation by constructing an IRAK-M knockdown airway epithelial cell model in vitro,in order to identify a new target for the treatment of COPD.Methods:Airway epithelial cell lines were transfected with IRAK-M siRNA to construct the IRAK-M knockdown airway epithelial cell model.Then the airway epithelial cells transfected with IRAK-M siRNA or negative control were stimulated with cigarette smoke extract(CSE),and the levels of downstream inflammatory factors were detected.We also evaluated the expression of phosphorylated proteins to further explore the potential regulatory mechanisms.All results were verified in LPS-induced airway epithelial cells.Results:The expression of IRAK-M was up-regulated by CSE and LPS stimulation in airway epithelial cells.Knockdown of IRAK-M can further promote the expression of inflammatory cytokines(IL-6,IL-8,TNF-α,IL-1β)and epithelial cell alarmin cytokines(TSLP,IL-33),as well as the C-X-C motif chemokine ligand 10(CXCL-10)induced by CSE and LPS in airway epithelial cells.Furthermore,the expression of phosphorylated NF-κB and phosphorylated P38 MAPK proteins in IRAK-M knockdown airway epithelial cells was significantly higher than that in the negative control group after CSE/LPS stimulation.Inhibiting NF-κB eliminated the statistical difference in IL-6 and IL-8 expression induced by CSE/LPS between the IRAK-M knockdown and negative control group,while inhibiting P38 MAPK had no significant effect on inflammatory cytokine secretion.Conclusion:IRAK-M can limit cigarette smoke-induced airway epithelial inflammation by inhibiting the excessive activation of NF-κB.Part 2:Role of IRAK-M in regulating biological behavior of lung fibroblasts and the potential molecular mechanisms investigationBackground and Objectives:The irreversible changes of airway remodeling pose a significant challenge for the treatment and management of COPD.Fibroblasts are highly active metabolic stromal cells that play an important role in subepithelial fibrosis and airway remodeling.The IRAK-M,a negative regulator of the TLR pathway,was found to be abundantly expressed in lung fibroblasts.However,there is little evidence on the direct effect of IRAK-M on lung fibroblasts phenotypes.Our study aims to explore the direct regulatory effect and related mechanisms of IRAK-M on cigarette smoke-induced lung fibroblast phenotype by constructing the IRAK-M knockdown lung fibroblast model in vitro,in order to find new targets for the prevention and treatment of airway remodeling in COPD.Methods:IRAK-M knockdown was performed by transfecting lung fibroblast cell lines with IRAK-M siRNA before challenging with CSE/LPS.The expression levels of fibrosisrelated proteins,as well as changes in proliferation,migration,and invasion events were evaluated.Furthermore,the expression of phosphorylated proteins was detected to explore the potential regulatory mechanisms of IRAK-M on lung fibroblast phenotype.All results were validated in LPS-induced lung fibroblasts.Results:1)CSE and LPS stimulation significantly up-regulated the expression of IRAKM in lung fibroblasts;2)Stimulation with CSE and LPS induced the expression of fibronectin,collagen I,matrix metalloproteinase 2,matrix metalloproteinase-9,and alphasmooth muscle actin in lung fibroblasts.Knockdown of IRAK-M further increased the expression of fibronectin,matrix metalloproteinase,and alpha-smooth muscle actin,while the expression of collagen I was downregulated;3)Knockdown of IRAK-M significantly promoted the proliferation,migration,and invasion of lung fibroblasts,and this effect was independent of CSE/LPS stimulation;4)The phosphorylation of P38 MAPK protein in lung fibroblasts stimulated with CSE/LPS was significantly higher in the IRAK-M knockdown group than that in the negative control group,and the enhanced proliferation,migration,and invasion ability of lung fibroblasts caused by IRAK-M knockdown could be significantly attenuated by inhibition of P38 MAPK;5)IRAK-M knockdown significantly down-regulated the mRNA and protein expression of an important antifibrotic cytokine,CXCL-10;6)IRAK-M knockdown significantly promoted the secretion of inflammatory factors in lung fibroblasts,and this effect was independent of CSE/LPS stimulation.Conclusion:IRAK-M was involved in airway remodeling by directly regulating the proliferation,migration,and invasion phenotypes of lung fibroblasts through the P38 MAPK pathway.Furthermore,IRAK-M has a direct impact on the expression of fibrosisrelated proteins,inflammatory and anti-fibrotic cytokines in lung fibroblasts.