The Mechanism Of TET2 Regulated Airway Epithelial Cell Ferroptosis In The Pathogenesis Of COPD

Objective:With an aging era coming,chronic obstructive pulmonary disease（COPD）has become a growing global burden of disease.Because of the chronic course and complex pathogenesis of COPD,the therapeutic options to cure are limited.In-depth investigation of the pathogenesis of COPD is an urgent need for the development of new therapeutic options.Cigarette smoking is the most common risk factor of COPD.Studies have shown that the recent named cell death（ferroptosis）characterized by iron depended lipid peroxidation,may be involved in the pathogenesis of smoking COPD.However,the mechanism remains unclear.Demethylase TET2（ten-eleven-translocation protein 2）-mediated demethylation plays an important role in cell death.Our group found that TET2 was downregulated in lung tissue of COPD patients,thus we would like to investigate the role of TET2 in cigarette smoke induced COPD.This study has four objectives.Firstly,to further validate the expression and distribution of TET2 in the lungs of COPD patients and cigarette smoke（CS）exposed mouse model.Secondly,to explore the role of TET2 in the pathogenesis of CS-induced COPD.Thirdly,to investigate the mechanism of TET2 on CS-induced ferroptosis in airway epithelial cells.At last,to observe whether the DNA methylation inhibitor 5-AZA combined with N-acetylcysteine has an enhanced protective effect on CS-exposed mouse model by antagonizing cellular ferroptosis.Methods:1.TET2 expression levels and distribution in lung tissues of COPD patients and CS-exposed mouse models were measured by immunohistochemistry（IHC）and Western-blot（WB）.2.Assessing the effect of TET2 knockout on lung pathological changes in CS-exposed mice:TET2 knockout mice and wild type（WT）mice were bred and were genotyped by agarose gel electrophoresis and exposed to room air（RA）or cigarette smoke（CS）for 6 months.They were divided into:wild-type mice with air-exposed group（WT RA）,TET2knockout mice with air-exposed group(TET2^-/-RA),wild-type mice with CS-exposed group（WT CS）and TET2 knockout mice with CS-exposed group(TET2^-/-CS).Mouse mortality was recorded during the experiment and survival analysis was performed on four groups to assess whether there was an effect of TET2 knockout on the survival of CS exposed mice.Emphysema of the lung of mice were assessed by Micro-CT.The pathomorphological changes in lung tissue such as emphysema and airway remodeling were assessed by Hematoxylin-Eosin（HE）stanning.MLI and DI index in emphysema,and the area of epithelial cell in perμm of basement membrane in the mouse airway were plotted.Collagen（fibrin）deposition and the small airway remodeling in mice were measured by Masson staining.The inflammatory cells in the bronchial airway lavage fluid（BALF）were counted and sorted to assess airway inflammation.3.To study the effect of TET2 knockout on ferroptosis in the lung tissue of mouse model after CS exposure,the mitochondrial morphology of mouse lung tissue was observed by transmission electronic microscope（TEM）.Besides,mouse lung tissue sections were stained with DAB-enhanced Prussian blue staining to assess the iron accumulation in the lung tissue of mice.Lipid peroxides in mouse lung tissue were detected by 4-HNE（4-hydroxynonenoic acid）staining.Total glutathione and oxidized glutathione disulfide（GSSG）,reduced glutathione（GSH）and GSSG ratio in mouse lung tissues were measured by GSH/GSSG detection kit.TET2and ferroptosis-related protein glutathione peroxidase 4（GPX4）expression in lung tissue were detected by western-blot.4.In vitro,we silenced or overexpressed the TET2 gene to investigate the effect of TET2 on CSE-induced ferroptosis.The bronchial epithelial cell line BEAS-2B were cultured.Firstly,BEAS-2B cells were transfected with TET2 si RNA for 48h,and then were treated with CSE or CSE combined with ferrostatin-1（Fer-1,inhibitor of ferroptosis）.For TET2overexpression,a plasmid containing the full-length of TET2 gene was constructed using the Crispr-cas9 technique.TET2 overexpressed plasmid were transfected into BEAS-2B cell and a TET2 stable expressing cell line were established and were then for the intervention of CSE or CSE combined with Fer-1.Cell morphological changes were observed under an inverted microscope.Cell viability was measured using CCK-8.Morphological changes of intracellular mitochondria were observed under TEM.GSH level was assessed by GSH/GSSG kit.Besides,the intracellular lipid peroxide（LPO）levels were detected by flow cytometry.To assess the changes of GPX4 and SLC7A11 in response to TET2modulation and CSE stimulation,GPX4 and SLC7A11 protein expression were detected by western-blot.5.To investigate the regulating mechanism of TET2 on GPX4expression.The m RNA expression of GPX4 in TET2-silenced or overexpressed cells were detected by RT-q PCR.The Cp G islands in the promoter of GPX4 gene were predicted on Methprimer and EBI Cp Gplot website.The promoter methylation of GPX4 were detected by MSP.6.To investigate the enhanced effect of DNA methylation inhibitors and antioxidants on the treatment of COPD,the methylation inhibitor of 5-AZA combined with antioxidants-NAC were applied to mouse with CS-exposure.The mice were divided into five groups:RA-exposed mice group,CS-exposed mice group,CS+NAC group,CS+5-AZA group,CS+NAC+5-AZA group.TEM was used to assess the morphology change of mitochondria in the lung tissue cells of mice.GSH and lipid peroxide levels of lung cells were detected by kit and flow cytometry,respectively.To assess the airway remodeling and emphysema of mice in each group,HE staining was applied and MLI,DI and the area of epithelial cell in perμm of basement membrane were plotted.Results:1.The expression of TET2 in the lung tissue of COPD patients,CS-exposed mice and CSE treated BEAS-2B cells.The IHC stanning and western-blot showed that TET2 was mainly expressed in the airway epithelial cells and was reduced in the lung tissues from COPD patients and CS-exposed mice.Besides,TET2 protein expression decreased in a CSE concentration dependent manner and appeared to be significantly reduced at 5%CSE concentration with CSE treatment in vitro.2.The role of TET2 deficiency in the development of CS induced COPD.（1）Survival analysis showed that all mice survived to the endpoint of the study,except TET2^-/-mice with CS exposure.（2）Micro-CT results showed that anteroposterior-to-transverse diameter of the thorax increased and lung translucency reduced in WT CS-exposed mice.TET2^-/-mice had an increased anteroposterior-to-transverse diameter ratio,reduced lung transparency and emphysema after CS exposure.（3）HE staining showed that CS-exposed mice presented significant emphysema with greater MLI and DI indices,compared with RA exposure.TET2^-/-mice showed the manifestation of emphysema with higher MLI and DI indices compared with WT group.And TET2^-/-CS mice showed higher MLI and DI indices compared with TET2^-/-RA and WT RA mice,and the most pronounced emphysema.（4）The area of epithelial cells perμm of basement membrane in the airways of mice revealed that the airways of CS-exposed mice showed local accumulation,disorganized arrangement,and thickening of epithelial cells.TET2^-/-mice with CS exposure showed more pronounced epithelial thickening of small airways compared to WT mice.（5）Masson staining showed that both WT and TET2^-/-mice showed significant collagen deposition under the airway epithelium after CS exposure compared to RA exposure.However,TET2^-/-CS mice showed more pronounced collagen deposition comparing to WT CS mice.（6）The number of total leukocytes,neutrophils and macrophages in BALF were significantly increased in WT and TET2^-/-mice after CS exposure.Compared with the WT CS group,the number of neutrophil in the BALF of TET2^-/-CS group was significantly increased,and with the increasing trend of total leukocytes and macrophages.3.The effect of TET2 on cigarette smoke-induced ferroptosis in airway epithelial cells（1）TEM results showed that CS-exposed mice showed significant morphological changes of ferroptosis,including accumulation of smaller mitochondrial volume,increased membrane density,fewer or disappeared mitochondrial ridges and rupture of the outer mitochondrial membrane,when compared with RA-exposed mice.TET2 knockout exacerbated mitochondrial morphology rupture under CS exposure.（2）DAB enhanced Prussian blue staining showed that CS exposure significantly increased iron accumulation in the airway epithelium of mouse lung tissue.Though,iron accumulation was observed in the airway epithelium in TET2^-/-mice than WT mice,there is no significance.TET2KO combined with CS exposure cannot further increase the iron level in the airway epithelium of lung tissue.（3）4-HNE staining showed that the lipid peroxide product 4-HNE in the lung tissue was significantly increased in the WT CS group,compared with the WT RA group.The content of 4-HNE in airway epithelial cells further increased in TET2 knockout mice with CS treatment.（4）There are no significant changes in GSH levels in the lung tissues of TET2^-/-and WT mice,while both GSH levels and GSH/GSSG ratio significantly decreased after CS exposure.When compared with WT CS group,GSH in the lung tissues decrease in TET2^-/-CS mice,while no significant change in GSH/GSSG ratio.（5）In the lung tissue of WT CS group mice,GPX4 protein expression was significantly decreased compared to WT RA group mice.The protein expression was significantly lower in lung tissues of TET2 knockout mice compared to WT mice.In addition,GPX4 protein expression was the lowest in lung tissue of TET2^-/-CS group mice,comparing to other groups.（6）CSE significantly reduced cell viability of BEAS-2B.Besides,TET2 silence further increased cell death in CSE treated cell.Interestingly,the addition of Fer-1 partially attenuated CSE-induced cytotoxicity,while independent of TET2 silence.（7）The morphological change of mitochondria in BEAS-2B cells were observed by TEM.Compared with the NC group,the intracellular mitochondria were smaller,the mitochondrial ridges disappeared and the outer membrane was ruptured,due to CSE treatment.si-TET2 exacerbated these changes in CSE treated cells.（8）GSH assay showed that TET2 silencing alone did not affect the GSH/GSSG ratio,comparing to the NC group.However,with CSE treatment,the GSH/GSSG ratio was significantly reduced in si-TET2group.（9）LPO levels assessed by flow cytometric were increased in si-TET2or CSE treatment,compared with the NC group in BEAS-2B cells.And intracellular LPO levels were aberrantly increased in si-TET2 and CSE group.（10）WB results showed that comparing to NC group,GPX4 protein expression was significantly down-regulated in si-TET2 group,but with no change of SLC7A11.（11）TET2 overexpression increased the cell viability under CSE by CCK8 assay.Besides,significant increase in cell viability were observed in TET2 overexpressed cells,stimulating with CSE or Fer-1.（12）Comparing to vector,TET2 stable overexpressed cells had no changes of mitochondria observed by TEM.However,in the CSE treated cells,the mitochondrial outer membrane ruptured,shrunken and the mitochondrial ridges disappeared.While the mitochondrial morphology change turns normal after TET2 overexpression.（13）The GSH/GSSG ratio was increase in the TET2 overexpression group compared with the control group,and the difference was statistically significant.TET2 increased the GSH/GSSG ratio after CSE treatment.（14）The LPO assessed by flow cytometry showed that the intracellular LPO level was significantly increased under CSE intervention,compared with the control group,while TET2 overexpression significantly decreased the intracellular LPO level.（15）The western-blot results showed that GPX4 expression were increased after TET2 overexpression,while there is no influence on the expression of SLC7A11.Besides,TET2 partly restored GPX4 expression in CSE treated cells.4.Mechanism of TET2 regulated ferroptosis in airway epithelial cells（1）The m RNA expression of GPX4 was decreased in TET2 silencing or CSE treated cells.Besides,si-TET2 further decreased GPX4 m RNA expression under CSE treatment.（2）TET2 overexpression enhanced the m RNA expression of GPX4and restored GPX4 m RNA expression after CSE treatment.（3）The expression of GPX4 m RNA were downregulated in the lung tissue of COPD patients and murine models.TET2 knockout reduced the m RNA level of GPX4 in the lung tissue of mice.（4）5-AZA enhanced the protein and m RNA expression of GPX4 and partly restored the GPX4 expression under CSE treatment.（5）Methylation prediction showed that Cp G islands were enriched in the promoter region of GPX4 gene.MSP results showed that the GPX4promoter region was methylated,and TET2 overexpression down-regulated GPX4 methylation.5.Effect of the co-administration of methylation inhibitors and antioxidants on cellular ferroptosis and CS-exposed emphysema.（1）5-AZA or NAC treatment increased GPX4 protein compared to the CS mice,while the GPX4 expression was significantly increased after5-AZA and NAC co-administration.（2）Only 5-AZA and NAC co-administration ameliorated the decrease in GSH/GSSG ratio and the increase in LPO caused by CS exposure.The5-AZA or NAC treatment alone cannot significantly increase GSH/GSSG ratio and inhibit LPO produce.（3）5-AZA and NAC co-administration alleviated the mitochondrial destruction of mouse lung tissue cells,comparing to CS group.（4）The co-administration of 5-AZA and NAC significantly improved emphysema and airway epithelial thickening in CS-exposed mice compared with 5-AZA or NAC alone.Conclusion:1.TET2 deficiency promotes the development of cigarette smoking-induced chronic obstructive pulmonary disease.2.TET2 regulated GPX4 promoter methylation involves in CSE induced airway epithelial cell ferroptosis.3.Demethylation compound combined with antioxidant antagonize ferroptosis to reduce smoking-induced emphysema.