Effects Of O₃ And DEP On Tight Junctions And Secretomics Of Airway Epithelial Cells

Outdoor air pollution has become a major public health problem worldwide and is an important cause of morbidity and mortality of chronic airway diseases.With global warming,ozone(O3)and particulate matter are the two main components of outdoor air pollutants in both developed and developing countries.Previou studies have shown that air pollutants can be tied to degradation by chemical or physical composition,inducing the expression of airway epithelial cell alarmins,such as IL-25,IL-33 and TSLP,and increasing the permeability of the epithelial barrier and inflammatory responses.At present,the specific biological effects of O3 and DEP exposure on airway epithelium have not been clarified.liquid-liquid interface culture was used mostly in previous studies on relevant mechanisms,which might change the physical or chemical properties of pollutants,and thus could not truly reflect the impact of pollutants intervention on respiratory tract.Air-liquid interface culture(ALI)is more suitable for medical and biological research because of its excellent repeatability and realizability in exposure conditions that are closer to the real physiological environment.Therefore,in this study,airway epithelial cells were exposed to O3 and DEP under the condition of gas-liquid culture(ALI),we aimed to explore the toxicity of O3 and DEP exposure to airway epithelial cells and tight intercellular junctions and alarmins,as well as effects of O3 and DEP exposure on secreted protein expression in airway epithelial cells.Part Ⅰ:Effects of O3 and DEP exposure on airway epithelial cell barrier functionPurpose:Based on the in vitro exposure system of gas-liquid culture,the effects of O3 and DEP exposure on airway epithelial cell barrier function and expression of alarmins were investigated.Methods:Calu-3 cells were cultured at the gas-liquid interface to establish the polarization model.The polarized epithelial cells were exposed to different concentrations of O3(0.5 ppm,1 ppm,2 ppm,4 ppm,1 hour)and DEP(100 mg/m3,200 mg/m3,300 mg/m3,400 mg/m3,1 hour)in the CULTEX? radial flow exposure system.After exposure,CCK8 and LDH methods were used to evaluate changes in cell activity under different exposure conditions,and cell resistance meter and FITC dextrin method were used to evaluate the barrier function of cell polarization model.Furthermore,q-PCR was used to evaluate the expression levels of alarmins(IL-25,IL-33,TSLP)and tight junction protein genes(ZO-1,Occludin,claudin1)in epithelial cells.Results:1.The TEER of Calu-3 cells with different inoculation densities showed the same trend.The polarization model with stable resistance value could be established when Calu3 cells were cultured in Tranwell seed plate with a density of 2×105/cm2 for 13 days with TEER>1000 Ω.cm2.2.In the CULTEX? radial flow exposure system,exposure to 2 ppm O3 and 300 mg/m3 DEP for 1 h did not cause a decrease in Calu-3 cell activity and increased LDH.3.2 ppm O3 and 300 mg/m3 DEP exposure for 1 hour could significantly reduce the transmembrane resistance(TEER)and increase the permeability of polarization model.4.2 ppm O3 and 300 mg/m3 DEP exposure for 1 hour could significantly decrease the expression level of tight junction protein ZO-1(1.00±0.02 vs 0.46±0.04,P<0.01,1.07±0.1 vs 0.49±0.05,P<0.01,respectively)and significantly increase the expression level of IL25(1.16±0.14 vs 6.87±0.62,P<0.01,1.02±0.18 vs 222.06±14.19,P<0.01,respectively),IL33(1.06±0.09 vs 9.68±0.63,P<0.01,1.09±0.45 vs 17.47±0.43,P<0.01,respectively)and TSLP(1.24±0.15 vs 2.12±0.43,P<0.05,1.03±0.23 vs 46.35±7.11,P<0.01,respectively)in airway epithelial cells.Conclusions:In this part,the cell polarization model of Calu-3 was established successfully.2 ppm O3 and 300 mg/m3 DEP exposure for 1 hour both damaged the airway epithelial cell barrier function and promoted the secretion of alarmins.Part Ⅱ:Effect of O3 and DEP exposure on secretory omics of airway epithelial cellsObjective:To investigate the effects of O3 and DEP exposure on secretory omics of airway epithelial cell.Methods:Human respiratory epithelial cell line Calu-3 was used to establish the polarization model of airway epithelial cells(forming airway barrier).Precise pollutant exposure of the polarization model was carried out based on CULTEX? radial flow exposure system.Cell culture medium of O3(2 ppm for 1 h)and DEP(300mg/m3 for 1 h)was collected for 24 hours after single exposure.Based on LC-MS/MS quantitative proteomics and Label free method,the expression of secretory proteome in the culture medium of airway epithelial cells after O3 and DEP exposure alone was analyzed.Results:1.The protein samples were qualified in quality control,and the protein abundance of 50 KDa was high.Based on Label free method,a total of 14986 peptides and 2274 proteins were identified under three different exposure conditions(filtered air group,O3 group and DEP group),and the data were qualified.2.Differential proteins were screened by the criterion of significant changes in protein expression abundance.Compared with filtered air group,there were 133 different proteins in O3 group,among which 98 were up-regulated and 35 were down-regulated.Compared with the filtered air group,there were 153 differentially expressed proteins in DEP group,including 86 up-regulated proteins and 67 down-regulated proteins.Compared with DEP group,there were 36 different proteins in O3 group,among which 26 were up-regulated and 10 were down-regulated.3.Functional classification of differential proteins were performed.Compared with the filtered air group,enrichment of O3 group differences in protein function mainly involved in the cell membrane and its membrane components,cytoskeleton cells,extracellular part and cell membrane complex,the function mainly relates to ferric iron ions,the substrate specificity of transporters activity,biological regulation,biological quality regulation,presenting antigen processing and biological processes,such as protein aggregation.The functional enrichment of differential proteins in DEP group are mainly involved in the activities of membrane and its membrane components,extracellular and its components and MHC Ⅱ protein complex,and their functions mainly involve the activity of protein kinase,the activity of serine endopeptidase inhibitor,the activity of peptidase inhibitor,cysteine peptidase and the binding of ferric ion.It is involved in stimulus response,immune response,cellular protein modification,antigen processing and presentation,and protein phosphorylation.Further,compared with DEP group,functional enrichment of differential proteins in O3 group are mainly involved in cytoplasmic proteins,cytoskeleton proteins and ribosomal proteins,and including tRNA binding,glucose 6-phosphate dehydrogenase activity,NADP binding,biliverdin reductase activity and eukaryotic initiation factor E4 binding.Mainly involved in single organism process,single organic somatic process,cell process and other biological processes.4.Enrichment analysis through KEGG pathway.Compared with the filtered air group,the first five pathways of differential protein enrichment in O3 group were Thl and Th2 cell differentiation,Staphylococcus aureus infection,HTLV-I infection,viral myocarditis and inflammatory bowel disease.The first 5 pathways of differential protein enrichment in DEP group were Th1 and Th2 cell differentiation,inflammatory bowel disease,rheumatoid arthritis,Staphylococcus aureus infection and Th17 cell differentiation.The first five pathways of differential protein enrichment in DEP group and O3 group were acute myeloid leukemia,2-oxycyclopentane carboxylic acid metabolism,osteoclast differentiation,endocrine resistance and phenylalanine,tyrosine and tryptophan biosynthesis and other signaling pathways.5.Combined with KEGG and String databases,protein interaction network analysis was performed on the differential proteins arrayed in the first five pathways.In O3 group,HLA-DRA protein,MHC class Ⅱ antigen,HLA-DQβ-chain protein,complement C5,ICAM-1,fibrinogen(y chain),calreticulin,actin interacted with various proteins.DEP group HLA-DRA,MHC CLASS Ⅱ antigen,HLA-DQβ-chain protein,complement C5,mitogen-activated protein kinase,Procathe Psin L interact with various proteins.Among the differential proteins in DEP group and O3 group,aspartate aminotransferase in cytoplasm,isocitrate dehydrogenase subunit α in mitochondria and mitogenactivated protein kinase 14 interacted with various proteins.This result may indicate the key proteins in the related pathway.Conclusions:Exposure to DEP and O3 alone can cause changes in the proteomics of airway epithelial cells,and they are involved in many pathways,among which the Thl and Th2 cell differentiation signaling pathways is the joint action of their pathways,and HLA-DRA,HLA-DQβ chain and MHC Ⅱ antigens are the key proteins of this pathway.