Background: Nuclear factor E2-related factor 2 (Nrf2) is involved in oxidative stress and lung inflammation and regulates the etiology of chronic obstructive pulmonary disease (COPD). Ferroptosis is characterized by the accumulation of lipid reactive oxygen species (ROS) via ferrous ion-dependent Fenton reactions and is involved in COPD. However, the role of Nrf2 in ferroptosis and its epigenetic regulation in the pathogenesis of COPD remain unclear. Methods: Ferroptosis was detected by 4-HNE, MDA, C11BODIPY, DCFH-DA, Peals’ staining and CCK-8 assays. qPCR and Western blotting were performed to examine the Nrf2 levels in peripheral lung tissues, primary epithelial cells collected from patients with COPD and subjects with normal pulmonary function (never-smoker [control-NS]; smoker [control-S]), and cigarette smoke extract (CSE)-treated human bronchial epithelial (HBE) cells. ELISA was used to quantify IL-8 and IL-1β levels. Methylation of the Nrf2 promoter was analyzed by bisulfite sequencing and pyrosequencing. Results: Ferroptosis was involved in COPD and glutathione peroxidase 4 (GPX4) expression was downregulated in the COPD group. Reactive oxygen species (ROS), lipid peroxides and MDA were increased, but GPX4 and SOD were exhausted in CSE-treated HBE cells. The production of IL-1β and IL-8 was promoted in HBE cells in response to CSE but could be reversed by the ferroptosis inhibitor fer-1. The Nrf2 level was significantly decreased in the COPD group compared with the control-S and control-NS groups. Increased Nrf2 expression enhanced GPX4 and SOD levels and inhibited ferroptosis and proinflammatory cytokines in the supernatant. Inhibition of GPX4 reversed the effect of Nrf2 overexpression and promoted ferroptosis. Two specific CpG sites within the Nrf2 promoter were hypermethylated in the COPD group. Similarly, CSE-treated HBE cells exhibited hypermethylation of the Nrf2 gene. Conclusion: Nrf2 expression was downregulated in the lungs of COPD patients due to hypermethylation of the Nrf2 promoter, inhibiting Nrf2/GPX4 and ferroptosis, which is related to the initiation and progression of COPD. Targeting Nrf2/GPX4 may inhibit ferroptosis, which could provide strategies to delay or treat COPD.

Background: Macrophage polarization plays an important role in the pathogenesis of COPD emphysema. Changes in macrophage polarization in COPD remain unclear, while polarization and ferroptosis are essential factors in its pathogenesis. Therefore, this study investigated the relationship between macrophage polarization and ferroptosis in COPD emphysema. Methods: We measured macrophage polarization and the levels of matrix metalloproteinases (MMPs) in the lung tissues of COPD patients and cigarette smoke (CS)-exposed mice. Flow cytometry was used to determine macrophage (THP-M cell) polarization changes. Ferroptosis was examined by FerroOrange, Perls’ DAB, C11-BODIPY and 4-HNE staining. Nuclear receptor coactivator 4 (NCOA4) was measured in the lung tissues of COPD patients and CS-exposed mice by western blotting. A cell study was performed to confirm the regulatory effect of NCOA4 on macrophage polarization. Results: Increased M2 macrophages and MMP9 and MMP12 levels were observed in COPD patients, CS-exposed mice and THP-M cells cocultured with CS extract (CSE)-treated human bronchial epithelial (HBE) cells. Increased NCOA4 levels and ferroptosis were confirmed in COPD. Treatment with NCOA4 siRNA and the ferroptosis inhibitor ferrostatin-1 revealed an association between ferroptosis and M2 macrophages. These findings support a role for NCOA4, which induces an increase in M2 macrophages, in the pathogenesis of COPD emphysema. Conclusion: In our study, CS led to the dominance of the M2 phenotype in COPD. We identified NCOA4 as a regulator of M2 macrophages and emphysema by mediating ferroptosis, which offers a new direction for research into COPD diagnostics and treatment.

Background: M2 polarized macrophages are involved in the occurrence and development of emphysema in COPD patients. However, the molecular mechanism of M2 macrophage polarization is still unclear. This study investigated the molecular mechanism of let-7 differentially expressed in bronchial epithelial cells of COPD patients participating in COPD emphysema by regulating the expression of IL-6 and inducing M2 polarization of alveolar macrophages (AM). Materials and Methods: We measured let-7c expression in human lung tissue, serum and the lung tissue of cigarette smoke (CS)-exposed mice by qRT‒PCR. We observed the M1/M2 AM polarization in the lungs of COPD patients and COPD model mice by immunofluorescence analysis. Western blotting was used to determine the expression of MMP9/12 in the lung tissue of COPD patients and CS-exposed mice. An in vitro experiment was performed to determine the molecular mechanism of let-7c-induced macrophage polarization. Results: Let-7c expression was downregulated in COPD patients, CS-exposed mice, and CS extract (CSE)-treated human bronchial epithelial (HBE) cells. AMs in COPD patients and CS-exposed mice were dominated by the M2 type, and the release of MMP9/12 was increased. In vitro, the transfection of mimics overexpressing let-7 or the use of tocilizumab to block signal transduction between HBE cells and macrophages inhibited the IL-6/STAT3 pathway. M2 macrophage polarization was inhibited, and MMP9/12 release was reduced. Conclusion: Our results indicate that CS decreased let-7c expression in HBE cells, and M2 AM polarization was dominant in COPD. In HBE cells, let-7c could inhibit M2 polarization of AMs through the IL-6/STAT3 pathway, providing potential diagnostic and therapeutic value for slowing COPD emphysema.

Background: This study aims to investigate the association between immune cells and the development of COPD, while providing a new method for the diagnosis of COPD according to the changes in immune microenvironment. Methods: In this study, the “CIBERSORT” algorithm was used to estimate the tissue infiltration of 22 types of immune cells in GSE20257 and GSE10006. The “limma” package was used for differentially expressed analysis. The key modules associated with vital immune cells were identified using WGCNA. GO and KEGG enrichment analysis revealed the biological functions of the candidate genes. Ultimately, a novel diagnostic prediction model was constructed via machine learning methods and multivariate logistic regression analysis based on GSE20257. Furthermore, we examined the stability of the model on one internal test set (GSE10006), three external test sets (GSE8545, GSE57148 and GSE76925), one single-cell transcriptome dataset (GSE167295), macrophages (THP-M cells) and lung tissue from COPD patients. Results: M0 macrophages (AUC > 0.7 in GSE20257 and GSE10006) were considered as the most important immune cells through exploring the immune microenvironment landscapes in COPD patients and healthy controls. The differentially expressed genes from GSE20257 and GSE10006 were divided into six and five modules via WGCNA, respectively. The green module in GSE20257 (cor = 0.41, P < 0.001) and the brown module in GSE10006 (cor = 0.67, P < 0.001) were highly correlated with M0 macrophages and were selected as key modules. Forty-one intersected genes obtained from two modules were primarily involved in regulation of cytokine production, regulation of innate immune response, specific granule, phagosome, lysosome, ferroptosis, and other biological processes. On the basis of the candidate genetic markers further characterized via the “Boruta” and “LASSO” algorithm for COPD, a diagnostic model comprising CLEC5A, FTL and SLC2A3 was constructed, which could accurately distinguish COPD patients from healthy controls in multiple datasets. GSE20257 as the training set has an AUC of 0.916. The AUCs of the internal test set and three external test sets were 0.873, 0.932, 0.675 and 0.688, respectively. Single-cell sequencing analysis suggested that CLEC5A, FTL and SLC2A3 were expressed in macrophages from COPD patients. The expressions of CLEC5A, FTL and SLC2A3 were up-regulated in THP-M cells and lung tissue from COPD patients. Conclusion: According to the variations of immune microenvironment in COPD patients, we constructed and validated a novel macrophage M0-associated diagnostic model with satisfactory predictive value. CLEC5A, FTL and SLC2A3 are expected to be promising targets of immunotherapy in COPD.

Background: Airway remodelling plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Epithelial–mesenchymal transition (EMT) is a significant process during the occurrence of airway remodelling. Increasing evidence suggests that glucose transporter 3 (GLUT3) is involved in the epithelial mesenchymal transition (EMT) process of various diseases. However, the role of GLUT3 in EMT in the airway epithelial cells of COPD patients remains unclear. Methods: We detected the levels of GLUT3 in the peripheral lung tissue of COPD patients and cigarette smoke (CS)-exposed mice. Two Gene Expression Omnibus GEO datasets were utilised to analyse GLUT3 gene expression profiles in COPD. Western blot and immunofluorescence were used to detect GLUT3 expression. In addition, we used the AAV9-GLUT3 inhibitor to reduce GLUT3 expression in the mice model. Masson’s staining and lung function measurement were used detect the collagen deposition and penh in the mice. A cell study was performed to confirm the regulatory effect of GLUT3. Inhibition of GLUT3 expression with siRNA, Western blot, and immunofluorescence were used to detect the expression of E-cadherin, N-cadherin, vimentin, p65, and ZEB1. Results: Based on the GEO data set analysis, GLUT3 expression in COPD patients was higher than in non-smokers. Moreover, GLUT3 was highly expressed in COPD patients, CS exposed mice, and BEAS-2B cells treated with CS extract (CSE). Further research revealed that down-regulation of GLUT3 significantly alleviated airway remodelling in vivo and in vitro. Lung function measurement showed that GLUT3 reduction reduced airway resistance in experimental COPD mice. Mechanistically, our study showed that reduction of GLUT3 inhibited CSE-induced EMT by down-regulating the NF-κB/ZEB1 pathway. Conclusion: We demonstrate that CS enhances the expression of GLUT3 in COPD and further confirm that GLUT3 may regulate airway remodelling in COPD through the NF-κB/ZEB1 pathway; these findings have potential value in the diagnosis and treatment of COPD. The down-regulation of GLUT3 significantly alleviated airway remodelling and reduced airway resistance in vivo. Our observations uncover a key role of GLUT3 in modulating airway remodelling and shed light on the development of GLUT3-targeted therapeutics for COPD.

Emphysema is a pathological mechanism of chronic obstructive pulmonary disease (COPD); however, its specific mechanism is unknown. Iron disorders are linked to ferroptosis, but the specific mechanism underlying iron homeostasis imbalance in alveolar epithelial cells in smoke-stimulated COPD emphysema is unclear. The present study investigated ferroptosis markers, lipid peroxidation, and Fe²⁺ levels in patients with COPD, smoke-exposed mice, and A549 cells. Techniques included H&E staining and lung function tests, western blotting/immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)/FerroOrange staining, and C11-BODIPY. Bioinformatics and in vitro experiments explored FTH1's role in COPD emphysema. We used AAV5-FTH1 plasmids to increase FTH1 expression in mice, CS exposed for 6 months, and assessed emphysema and ferroptosis-related indicators in mice. Co-immunoprecipitation was used to detect the binding of FTH1 with O-GlcNAcase (OGA), O-GlcNAc, and nuclear receptor coactivator 4. TMG, FTH1, and SLC40A1 plasmids were added in A549 cells, exposed in 5 % CSE, and indicators of ferroptosis were detected. Ferroptosis and ion disorders were major causes of COPD emphysema in vitro and in vivo. We determined that FTH1 plays a key role in the steady-state imbalance of iron ions and ferroptosis in A549 cells exposed to cigarette smoke (CS). FTH1 expression was high in human lung tissue. In A549 cells, O-GlcNAc, which can bind with FTH1, decreased. The addition of TMG reversed the ferroptosis. Finally, FTH1 prevents COPD formation in mice. CS increases OGA expression in alveolar epithelial cells and causes iron metabolism disorders, which leads to ferroptosis and may facilitate the formation of COPD emphysema.