Airway Epithelial-mesenchymal Transition Research Articles

Scutellarin Alleviates Ovalbumin-Induced Airway Remodeling in Mice and TGF-β-Induced Pro-fibrotic Phenotype in Human Bronchial Epithelial Cells via MAPK and Smad2/3 Signaling Pathways.

Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness (AHR), inflammation, and remodeling. Epithelial-mesenchymal transition (EMT) is an essential player in these alterations. Scutellarin is isolated from Erigeron breviscapus. Its vascular relaxative, myocardial protective, and anti-inflammatory effects have been well established. This study was designed to detect the biological roles of scutellarin in asthma and its related mechanisms. The asthma-like conditions were induced by ovalbumin challenges. The airway resistance and dynamic compliance were recorded as the results of AHR. Bronchoalveolar lavage fluid (BALF) was collected and processed for differential cell counting. Hematoxylin and eosin staining, periodic acid-Schiff staining, and Masson staining were conducted to examine histopathological changes. The levels of asthma-related cytokines were measured by enzyme-linked immunosorbent assay. For in vitro analysis, the 16HBE cells were stimulated with 10 ng/mL transforming growth beta-1 (TGF-β1). Cell migration was estimated by Transwell assays and wound healing assays. E-cadherin, N-cadherin, and α-smooth muscle actin (α-SMA) were analyzed by western blotting, real-time quantitative polymerase chain reaction, immunofluorescence staining, and immunohistochemistry staining. The underlying mechanisms of the mitogen-activated protein kinase (MAPK) and Smad pathways were investigated by western blotting. In an ovalbumin-induced asthmatic mouse model, scutellarin suppressed inflammation and inflammatory cell infiltration into the lungs and attenuated AHR and airway remodeling. Additionally, scutellarin inhibited airway EMT (upregulated E-cadherin level and downregulated N-cadherin and α-SMA) in ovalbumin-challenged asthmatic mice. For in vitro analysis, scutellarin prevented the TGF-β1-induced migration and EMT in 16HBE cells. Mechanistically, scutellarin inhibits the phosphorylation of Smad2, Smad3, ERK, JNK, and p38 in vitro and in vivo. In conclusion, scutellarin can inactivate the Smad/MAPK pathways to suppress the TGF-β1-stimulated epithelial fibrosis and EMT and relieve airway inflammation and remodeling in asthma. This study provides a potential therapeutic strategy for asthma.

DPP4 Regulates the Th17/IL-17 Axis and Accelerates Epithelial Mesenchymal Transition to Promote Ovalbumin-Induced Asthma in Female C57BL/6J Mice.

Dipeptidyl peptidase-4 (DPP4) is a transmembrane glycoprotein, prevalent across a variety of tissues and cells and can be foundin a solubilised in peripheral blood. This paper aims at determining the role of sCD26/sDPP4 in Th17 cell polarization and airway epithelial cell to epithelial mesenchymal transition (EMT) in asthma. Female C57BL/6J mice were treated with ovalbumin to constructed asthma mice. The CD4+ T cell, and bronchial epithelial cells (BECs) were purified from the spleens and bronchus of mice. The KRT8 expression in BECs were identified by immunofluorescence (IF). Th17 cells were differentiated from a CD4+ T cell. Flow cytometry was usewd to identify and calculate the Th17 and Treg cells. Mice woth asthma were treated by DPP4 overexpressing lentivirus or DPP4 inhibitor. Histopathological modifications were assessed by hematoxylin-eosin (HE), periodic acid Schiff (PAS), and Masson staining. The total number of leucocytes was detected using a hemocytometer. For detection, quantitative Real-time PCR (qRT-PCR), western blotting (WB), and IF were used to evaluate the expression of E-cadherin and alpha-smooth muscle actin (α-SMA). Enzyme-linked immunosorbent assay (ELISA) was performed to analyze the DPP4, IL-4, IL-5, IL-13 and IL-17 levels. The findings suggest that sCD26/sDPP4 promote CD4+ T cells differentiation into Th17 cells in a depending on the applied dose. sCD26/sDPP4 up-regulated the expression of α-SMA and down-regulated the expression of E-cadherin in TGF-β1-induced mouse BECs, which was reversed by DPP4 inhibitor. Co-culture induced a synergic effect between Th17 cells and sCD26/sDPP4 on the formation of airway EMT in BECs. Furthermore, DPP4 inhibitor prevented lung-bronchial inflammatory infiltration, mucus secretion, goblet cell hyperplasia and collagen deposition in asthma mice. Meanwhile, DPP4 inhibitor decreased the levels of DPP4, IL-4, IL-5, IL-13, IL-17 and increased the total number of leukocytes in bronchoalveolar lavage fluid of asthma mice. In addition, DPP4 inhibitor also inhibited airway EMT and Th17 cell polarization in asthma mice. The results in this paper show that up-regulation of DPP4 enabled airway inflammation and airway remodeling in asthmatic mice by modulating the Th17/IL-17 axis and accelerating the airway EMT, which isa therapeutic target in asthma.

Semaphorin 7a aggravates TGF-β1-induced airway EMT through the FAK/ERK1/2 signaling pathway in asthma.

TGF-β1 can induce epithelial-mesenchymal transition (EMT) in primary airway epithelial cells (AECs). Semaphorin7A (Sema7a) plays a crucial role in regulating immune responses and initiating and maintaining transforming growth factor β1 TGF-β1-induced fibrosis. To determine the expression of Sema7a, in serum isolated from asthmatics and non-asthmatics, the role of Sema7a in TGF-β1 induced proliferation, migration and airway EMT in human bronchial epithelial cells (HBECs) in vitro. The concentrations of Sema7a in serum of asthmatic patients was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of Sema7a and integrin-β1 were examined using conventional western blotting and real-time quantitative PCR (RT-PCR). Interaction between the Sema7a and Integrin-β1 was detected using the Integrin-β1 blocking antibody (GLPG0187). The changes in EMT indicators were performed by western blotting and immunofluorescence, as well as the expression levels of phosphorylated Focal-adhesion kinase (FAK) and Extracellular-signal-regulated kinase1/2 (ERK1/2) were analyzed by western blot and their mRNA expression was determined by RT-PCR. We described the first differentially expressed protein of sema7a, in patients with diagnosed bronchial asthma were significantly higher than those of healthy persons (P<0.05). Western blotting and RT-PCR showed that Sema7a and Integrin-β1 expression were significantly increased in lung tissue from the ovalbumin (OVA)-induced asthma model. GLPG0187 inhibited TGF-β1-mediated HBECs EMT, proliferation and migration, which was associated with Focal-adhesion kinase (FAK) and Extracellular-signal-regulated kinase1/2 (ERK1/2) phosphorylation. Sema7a may play an important role in asthma airway remodeling by inducing EMT. Therefore, new therapeutic approaches for the treatment of chronic asthma, could be aided by the development of agents that target the Sema7a.

Proline is increased in allergic asthma and promotes airway remodeling.

Proline and its synthesis enzyme pyrroline-5-carboxylate reductase 1 (PYCR1) are implicated in epithelial-mesenchymal transition (EMT), yet how proline and PYCR1 function in allergic asthmatic airway remodeling via EMT has not yet been addressed to our knowledge. In the present study, increased levels of plasma proline and PYCR1 were observed in patients with asthma. Similarly, proline and PYCR1 in lung tissues were high in a murine allergic asthma model induced by house dust mites (HDMs). Pycr1 knockout decreased proline in lung tissues, with reduced airway remodeling and EMT. Mechanistically, loss of Pycr1 restrained HDM-induced EMT by modulating mitochondrial fission, metabolic reprogramming, and the AKT/mTORC1 and WNT3a/β-catenin signaling pathways in airway epithelial cells. Therapeutic inhibition of PYCR1 in wild-type mice disrupted HDM-induced airway inflammation and remodeling. Deprivation of exogenous proline relieved HDM-induced airway remodeling to some extent. Collectively, this study illuminates that proline and PYCR1 involved with airway remodeling in allergic asthma could be viable targets for asthma treatment.

TMT-based quantitative proteomics revealed protective efficacy of Icariside II against airway inflammation and remodeling via inhibiting LAMP2, CTSD and CTSS expression in OVA-induced chronic asthma mice

BackgroundAsthma is a chronic inflammatory disorder in airways with typical pathologic features of airflow limitation, airway inflammation and remodeling. Icariside II (IS), derived from herbal medicine Herba Epimedii, exerts an anti-inflammatory property. However, underlying mechanisms with specifically targeted molecular expression by IS in asthma have not been fully understood, and whether IS could inhibit remodeling and EMT still remains unclear. PurposeThe study aimed to clarify therapeutic efficacy of IS for attenuating airway inflammation and remodeling in asthma, and illustrate IS-regulated specific pathway and target proteins through TMT-based quantitative proteomics. Study design and methodsMurine model of chronic asthma was constructed with ovalbumin (OVA) sensitization and then challenge for 8 weeks. Pulmonary function, leukocyte count in bronchoalveolar lavage fluid (BALF), lung histopathology, inflammatory and fibrotic cytokines, and markers of epithelial-mesenchymal transition (EMT) were evaluated. TMT-based quantitative proteomics were performed on lung tissues to explore IS-regulated proteins. ResultsIS contributed to alleviative airway hyperresponsiveness (AHR) evidenced by declined RL and increased Cdyn. After IS treatment, we observed a remarked down-regulation of leukocyte count, inflammatory cytokines in BALF, and peribronchial inflammation infiltration. Goblet cell hyperplasia, mucus secretion and peribronchial collagen deposition were attenuated, with the level of TGF-β and MMP-9 in BALF declined. Furthermore, IS induced a rise of Occludin and E-cadherin and a decline of N-cadherin and α-SMA in lung tissues. These results proved the protective property of IS against airway inflammation, remodeling and EMT. To further investigate underlying mechanisms of IS in asthma treatment, TMT-based quantitative proteomics were performed and 102 overlapped DEPs regulated by IS were identified. KEGG enrichment exhibited these DEPs were enriched in lysosome, phagosome and autophagy, in which LAMP2, CTSD and CTSS were common DEPs. WB, q-PCR and IHC results proofed expressional alteration of these proteins. Besides, IS could decrease Beclin-1 and LC3B expression with increasing p62 expression thus inhibiting autophagy. ConclusionsThe study demonstrated IS could ameliorate AHR, airway inflammation, remodeling and EMT in OVA-induced chronic asthma mice. Our research was the first to reveal that inhibition of LAMP2, CTSD and CTSS expression in autophagy contributed to the therapeutic efficacy of IS to asthma.

DOCK2 Promotes Asthma Development by Eliciting Airway Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) contributes to airway remodeling, a predominant feature of asthma. DOCK2 (dedicator of cytokinesis 2) is an innate immune signaling molecule involved in vascular remodeling. However, it is unknown if DOCK2 plays a role in airway remodeling during asthma development. In this study, we found that DOCK2 is highly induced in both normal human bronchial epithelial cells treated with house dust mite (HDM) extract and human asthmatic airway epithelium. DOCK2 is also upregulated by TGF-β1 (transforming growth factor β1) during EMT of human bronchial epithelial cells. Importantly, knockdown of DOCK2 inhibits, and overexpression of DOCK2 promotes, TGF-β1-induced EMT. Consistently, DOCK2 deficiency suppresses the EMT of airway epithelium, attenuates the subepithelial fibrosis, and improves pulmonary function in HDM-induced asthmatic lungs. These data suggest that DOCK2 plays an important role in EMT and asthma development. Mechanistically, DOCK2 interacts with transcription factor FoxM1 (forkhead box M1), which enhances FoxM1 binding to mesenchymal marker gene promoters and further promotes mesenchymal marker gene transcription and expression, leading to EMT. Taken together, our study identifies DOCK2 as a novel regulator for airway EMT in an HDM-induced asthma model, thus providing a potential therapeutic target for treatment of asthma.

PKM2 regulates cigarette smoke-induced airway inflammation and epithelial-to-mesenchymal transition via modulating PINK1/Parkin-mediated mitophagy

Cigarette smoke (CS) mediates inflammation and epithelial-mesenchymal transition (EMT) in bronchial epithelial cells, contributing to airway remodeling in chronic obstructive pulmonary disease (COPD). Cross-talk between metabolic pathways and cell signaling has emerged as an important focus of research in the field of inflammation. Here, we established in vitro and in vivo models of CS-induced COPD to elucidate the role of pyruvate kinase M2 (PKM2), a glycolytic enzyme, in CS-induced airway remodeling. Exposure to CS significantly increased PKM2 expression in lung tissues of C57BL/6 mice and BEAS-2B cells, which positively related to the levels of airway inflammation and EMT. Administering PKM2 inhibitor shikonin attenuated CS-induced airway inflammation and EMT process. Moreover, knockdown of PKM2 by small-interfering RNA (siRNA) decreased the release of TNF-α and IL-8, ROS and reversed the CS extract (CSE)-induced changes of N-cadherin and E-cadherin in BEAS-2B cells. In CSE-treated cells, we also observed enhancement of PINK1/Parkin-mediated mitophagy, which were decreased by PKM2 siRNA. Furthermore, pretreatment with mitophagy inducer CCCP before CSE stimulation led to increased expressions of both nuclear and cytosolic PKM2, accompanied by reduction of TGF-β-induced factor homeobox 2 (TGIF2), a repressor of TGF-β1/smad pathway and EMT, while PKM2 knockdown restored the expression of TGIF2. Our results imply that CS induces PKM2 upregulation in airway epithelial cells, acting in synergism with PINK/Parkin-mediated mitophagy, which may initiate and exaggerate airway inflammation and EMT process. Further studies will be required to elucidate more molecular details and other pathways by which PKM2-mitophagy signaling regulates the effector function of airway epithelial.

Hydrogen Sulfide Inhibits Bronchial Epithelial Cell Epithelial Mesenchymal Transition Through Regulating Endoplasm Reticulum Stress.

Epithelial mesenchymal transition (EMT) is a contributing factor in remodeling events of chronic obstructive pulmonary disease (COPD). Hydrogen sulfide (H2S) has been implicated in the pathogenesis of COPD, but the effect of H2S in regulating EMT and the underlying mechanisms is not clear. In this study, we assessed endoplasmic reticulum (ER) stress markers, EMT markers and associated signal molecules in rat lungs, bronchial epithelial cells, and human peripheral lung tissues to investigate the effect of H2S in regulating EMT and the underlying mechanisms. We found that EMT and ER stress occurred in lung epithelial cells, especially in the bronchial epithelial cells of smokers and COPD patients. In cigarette smoke (CS)-exposed rats, intraperitoneal injection of NaHS significantly alleviated CS-induced lung tissue damage, small airway fibrosis, ER stress, and EMT, while intraperitoneal injection of propargylglycine (cystathionine-gamma-lyase inhibitor) aggravated these effects induced by CS. In the nicotine-exposed 16HBE cells, an appropriate concentration of H2S donor not only inhibited nicotine-induced ER stress, but also inhibited nicotine-induced enhancement of cell migration ability and EMT. ER stress nonspecific inhibitors taurine and 4-phenyl butyric acid also inhibited nicotine-induced enhancement of cell migration ability and EMT. Both H2S and inositol-requiring enzyme 1 (IRE1) activation inhibitor 4μ8C inhibited nicotine-induced activation of IRE1, Smad2/3 and EMT. These results suggest that H2S inhibits CS- or nicotine-induced ER stress and EMT in bronchial epithelial cells and alleviates CS-induced lung tissue damage and small airway fibrosis. The IRE1 signal pathway and Smad2/3 may be responsible for the inhibitory effect of H2S.

IL-27 attenuates airway inflammation and epithelial-mesenchymal transition in allergic asthmatic mice possibly via the RhoA/ROCK signalling pathway

Asthma is an airway disease characterized by airflow limitation and various additional clinical manifestations. Repeated inflammatory stimulation of the airways leads to epithelial-mesenchymal transition (EMT) which aggravates subepithelial fibrosis during the process of airway remodelling and enhances resistance to corticosteroids and bronchodilators in refractory asthma. There is growing evidence that IL-27 modulates airway remodelling, however, the molecular mechanisms involving IL-27 and EMT are poorly understood. The objective of this study was to investigate the effects of IL-27 on ovalbumin (OVA)-challenged asthmatic mice in vivo and TGF-β1-induced EMT in 16HBE cells in vitro. Airway inflammation, mucus secretion, and collagen deposition were analysed by conventional pathological techniques. The ratio of Th17 and Th9 cells in the spleen of mice was measured using flow cytometry, ELISA was performed for cytokine analysis to identify EMT-related molecules and signalling pathways, and other molecular and cellular techniques were used to explore the functional mechanism involving IL-27 and EMT. Airway inflammation in asthmatic mice was significantly alleviated by IL-27, with downregulation of RhoA and ROCK, upregulation of E-cadherin, and a decrease of vimentin and α-SMA expression, compared to asthmatic mice. Moreover, the frequency of Th17 and Th9 cells in the spleen of asthmatic mice decreased following treatment with IL-27. In TGF-β1-induced 16HBE cells, the addition of IL-27 was shown to inhibit EMT, based on the expression of E-cadherin, vimentin, and α-SMA. Intranasal administration of IL-27 attenuates airway inflammation and EMT in a murine model of allergic asthma possibly by downregulating the RhoA/ROCK signalling pathway.

Increased myofibroblasts in the small airways, and relationship to remodelling and functional changes in smokers and COPD patients: potential role of epithelial–mesenchymal transition

IntroductionPrevious reports have shown epithelial–mesenchymal transition (EMT) as an active process that contributes to small airway fibrotic pathology. Myofibroblasts are highly active pro-fibrotic cells that secrete excessive and altered extracellular matrix (ECM). Here we relate small airway myofibroblast presence with airway remodelling, physiology and EMT activity in smokers and COPD patients.MethodsLung resections from nonsmoker controls, normal lung function smokers and COPD current and ex-smokers were stained with anti-human α-smooth muscle actin (SMA), collagen 1 and fibronectin. αSMA+ cells were computed in reticular basement membrane (Rbm), lamina propria and adventitia and presented per mm of Rbm and mm2 of lamina propria. Collagen-1 and fibronectin are presented as a percentage change from normal. All analyses including airway thickness were measured using Image-pro-plus 7.0.ResultsWe found an increase in subepithelial lamina propria (especially) and adventitia thickness in all pathological groups compared to nonsmoker controls. Increases in αSMA+ myofibroblasts were observed in subepithelial Rbm, lamina propria and adventitia in both the smoker and COPD groups compared to nonsmoker controls. Furthermore, the increase in the myofibroblast population in the lamina propria was strongly associated with decrease in lung function, lamina propria thickening, increase in ECM protein deposition, and finally EMT activity in epithelial cells.ConclusionsThis is the first systematic characterisation of small airway myofibroblasts in COPD based on their localisation, with statistically significant correlations between them and other pan-airway structural, lung function and ECM protein changes. Finally, we suggest that EMT may be involved in such changes.

N-myc-interactor mediates microbiome induced epithelial to mesenchymal transition and is associated with chronic lung allograft dysfunction

Recent evidence suggests a role for lung microbiome in occurrence of chronic lung allograft dysfunction (CLAD). However, the mechanisms linking the microbiome to CLAD are poorly delineated. We investigated a possible mechanism involved in microbial modulation of mucosal response leading to CLAD with the hypothesis that a Proteobacteria dominant lung microbiome would inhibit N-myc-interactor (NMI) expression and induce epithelial to mesenchymal transition (EMT). Explant CLAD, non-CLAD, and healthy nontransplant lung tissue were collected, as well as bronchoalveolar lavage from 14 CLAD and matched non-CLAD subjects, which were followed by 16S rRNA amplicon sequencing and quantitative polymerase chain reaction (PCR) analysis. Pseudomonas aeruginosa (PsA) or PsA-lipopolysaccharide was cocultured with primary human bronchial epithelial cells (PBEC). Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate NMI expression and EMT in explants and in PsA-exposed PBECs. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of EMT regulator NMI. 16S rRNA amplicon analyses revealed that CLAD patients have a higher abundance of phyla Proteobacteria and reduced abundance of the phyla Bacteroidetes. At the genera level, CLAD subjects had an increased abundance of genera Pseudomonas and reduced Prevotella. Human CLAD airway cells showed a downregulation of the N-myc-interactor gene and presence of EMT. Furthermore, exposure of human primary bronchial epithelial cells to PsA resulted in downregulation of NMI and induction of an EMT phenotype while NMI upregulation resulted in attenuation of this PsA-induced EMT response. CLAD is associated with increased bacterial biomass and a Proteobacteria enriched airway microbiome and EMT. Proteobacteria such as PsA induces EMT in human bronchial epithelial cells via NMI, demonstrating a newly uncovered mechanism by which the microbiome induces cellular metaplasia.

Cathelicidin induces epithelial-mesenchymal transition to promote airway remodeling in smoking-related chronic obstructive pulmonary disease

BackgroundEpithelial-mesenchymal transition (EMT) is an important characteristic in the remodeling of airways that occurs in chronic obstructive pulmonary disease (COPD). Cigarette smoke is a potential driving factor of this EMT in COPD. However, the mechanisms by which cigarette smoke induce EMT remain uncertain. Cathelicidin has been implicated as a causal factor of airway inflammation and mucus hypersecretion in smoking-related COPD. This study aimed to investigate whether cathelicidin induces EMT to promote airway remodeling in this disease.MethodsHuman lung tissue was collected from smokers with COPD and smokers without COPD. The EMT markers E-cadherin and vimentin were examined by immunohistochemistry. Mouse models of COPD were established by taking mice with airway cathelin-related antimicrobial peptide (CRAMP), the murine homologue of cathelicidin, either upregulated or downregulated by intranasal introduction of lentiviral vectors and then exposing them to cigarette smoke. E-cadherin and vimentin expression in the airways of the model mice was examined using immunofluorescence. Tumor necrosis factor alpha (TNF-α) converting enzyme (TACE), transforming growth factor alpha (TGF-α), and epidermal growth factor receptor (EGFR) expression was analyzed by Western blot. Additionally, NCI-H292 human airway epithelial cells, both with and without cathelicidin downregulation, were stimulated with cigarette smoke extract (CSE) and LL-37 synthetic peptide, a bioactive fragment of cathelicidin. This was done to confirm that the TACE/TGF-α/EGFR signaling pathway is activated in humans exposed to cigarette smoke.ResultsSignificant EMT was found in the small airways of smokers both with and without COPD, as well as in the airways of COPD model mice. Downregulation of CRAMP in COPD mice, however, ameliorated airway EMT induced by cigarette smoke. Conversely, upregulation of CRAMP enhanced airway EMT in vivo; TACE, TGF-α, and EGFR were found to be involved in this process. In vitro, EMT induced by CSE and LL-37 was inhibited by blocking TACE, TGF-α, and EGFR expression.ConclusionsCathelicidin promotes airway EMT by activating the TACE/TGF-α/EGFR signaling pathway. This mediates smoking-induced airway remodeling in the pathogenesis of COPD.

MSCs reduce airway remodeling in the lungs of asthmatic rats through the Wnt/β-catenin signaling pathway.

Asthma is a chronic pulmonary inflammatory disease characterized by excessive infiltration of leukocytes into the respiratory tract. We explored the underlying mechanisms of mesenchymal stem cells (MSCs) in the treatment of allergic asthma using a rat model. The rats were sensitized with ovalbumin (OVA) and an aluminium hydroxide emulsion, which were injected intraperitoneally, and then the sensitized rats were challenged with aerosolized OVA. Before the allergen challenge, the model rats were injected with MSCs and MSC-derived exosomes. At the same time, 2 out of the 6 groups of rats were injected with BML-284, a Wnt agonist. The degree of airway inflammation was determined by bronchoalveolar lavage fluid (BALF) and haematoxylin and eosin (H&E) staining; the degree of airway remodelling was assessed by Masson staining; Western blotting (WB) and real-time polymerase chain reaction (PCR) were performed to evaluate Wnt/β-catenin signalling pathway-related factors and the expression of epithelial-mesenchymal transition (EMT)-related proteins in lung tissues. We showed that among the rats that were sensitized and challenged with OVA, the injection of MSCs and MSC-derived exosomes significantly reduced the total number of cells and the number of immune cells in BALF, proliferation of goblet cells and collagen deposition. Moreover, the number of BALF cells and collagen deposition increased significantly after the injection of BML-284. WB and real-time PCR showed that MSCs and MSC-derived exosomes significantly inhibited airway remodelling and EMT by restricting the Wnt/β-catenin signalling pathway, while additional injection of BML-284 suppressed the effects of MSCs and their exosomes, increased the EMT of the airway epithelium and exacerbated airway remodelling. MSCs inhibit chronic allergic inflammation of the airway and reduce airway remodelling and EMT of the airway epithelium in the lungs of asthmatic rats. This process is partly attributed to the inhibition of the Wnt/β-catenin signalling pathway by MSC-derived exosomes.

The roles of dipeptidyl peptidase-4 and its inhibitors in the regulation of airway epithelial-mesenchymal transition

Objective: Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a transmembrane glycoprotein with peptidase activity expressed on epithelial cells and some immune cells. It also occurs as a soluble form. Studies have revealed that the expression level of lymphocyte sCD26/sDPP4 was elevated in the asthmatic patients. Airway remodeling increases in asthma severity and these structural changes include, amongst others, the loss of epithelial integrity because of cell shedding, goblet cell hyperplasia, destruction of ciliated cells, and EMT. So we try to find whether sCD26/sDPP4 has a role in pathological/dysregulated transition from bronchial epithelial cells into fibroblasts cells in response to TGFβ1 exposure in vitro. Therefore, our purpose in the present work was to identify the role of sCD26/sDPP4 in airway EMT regulation. Methods: The EMT cell model was established based on human 16HBE cells. The effects of sCD26/sDPP4 and its inhibitors on airway EMT and that of sCD26/sDPP4 on Th17/IL-17 and its role in airway EMT were investigated in vitro. Results: The mRNA and protein level of E-Cadherin decreased after the treatment of TGF-β1 in 16HBE cells, while α-SMA was up-regulated. The level of E-Cadherin was significantly down-regulated after the sCD26/sDPP4 stimulation, and that of α-SMA was dramatically elevated. DPP4 inhibitors promoted the level of E-cadherin and inhibited that of α-SMA. Additionally, in the DPP4-treated IL-17 cells group, E-Cadherin was markedly down-regulated at the mRNA and protein level, while α-SMA was reversely up-regulated. Conclusion: The TGF-β1-induced EMT of human bronchial epithelial cells could be promoted by sCD26/sDPP4. The suppression of EMT in human bronchial epithelial cells was achieved by DPP4 inhibitor, and the TGF-β1-mediated EMT of human airway cells was promoted by the synergy of IL-17 and sCD26/sDPP4 in vitro.

Gammaproteobacteria Dominant Lung Microbiome Induces Epithelial to Mesenchymal Transition and is Associated with CLAD

Recent evidence suggests a role for lung microbiome in occurrence of chronic lung allograft dysfunction (CLAD). However, the mechanisms linking microbiome to CLAD are not delineated. We investigated the mechanisms involved in microbial modulation of mucosal response leading to CLAD. We hypothesized that a Gammaproteobacteria dominant lung microbiome induces epithelial to mesenchymal transition (EMT) and correlates with CLAD. Explant CLAD and control lungs were collected. BAL was collected from 10 CLAD and matched non-CLAD subjects and 16S rRNA microbiome sequencing performed. 105CFU Pseudomonas aeruginosa (PA) was co-cultured with human bronchial epithelial cells (PBEC) (1:1) in RPMI media for 24 hours. Western blot and qRT-PCR was performed to evaluate EMT in explants and in PA exposed PBECs. These experiments were repeated after siRNA downregulation and upregulation (plasmid vector) of EMT regulator gene n-myc and stat interactor (NMI). Gene/protein expression of mesenchymal markers vimentin (V), α-SMA (A), n-cadherin (N) were elevated and epithelial marker e-cadherin (E), NMI significantly reduced in explant CLAD compared to controls. 16S rRNA analysis of CLAD BAL showed reduced bacterial diversity (Shannon index, p=0.01), increased bacterial biomass (16S gene copy/ml BAL, P=0.04) and a predominance of class Gammaproteobacteria (p=0.03) and genus Pseudomonas (p=0.01) compared to matched non-clad controls. PBEC exposure to PA resulted in increased expression of V, N, A and significant reduction in E suggesting EMT (Fig 1). Expression of NMI was significantly reduced in PA exposed PBECs. PA exposure in NMI overexpressed PBECs attenuated EMT and in NMI downregulated PBECs augmented EMT. CLAD is associated with reduced bacterial diversity, increased bacterial biomass, Gammaproteobacteria dominant airway microbiome and EMT. PA induces EMT in human bronchial epithelial cells. NMI is a critical regulator of PSA induced airway EMT.

Dual role of RACK1 in airway epithelial mesenchymal transition and apoptosis.

Airway epithelial apoptosis and epithelial mesenchymal transition (EMT) are two crucial components of asthma pathogenesis, concomitantly mediated by TGF‐β1. RACK1 is the downstream target gene of TGF‐β1 shown to enhancement in asthma mice in our previous study. Balb/c mice were sensitized twice and challenged with OVA every day for 7 days. Transformed human bronchial epithelial cells, BEAS‐2B cells were cultured and exposed to recombinant soluble human TGF‐β1 to induced apoptosis (30 ng/mL, 72 hours) and EMT (10 ng/mL, 48 hours) in vitro, respectively. siRNA and pharmacological inhibitors were used to evaluate the regulation of RACK1 protein in apoptosis and EMT. Western blotting analysis and immunostaining were used to detect the protein expressions in vivo and in vitro. Our data showed that RACK1 protein levels were significantly increased in OVA‐challenged mice, as well as TGF‐β1‐induced apoptosis and EMT of BEAS‐2B cells. Knockdown of RACK1 (siRACK1) significantly inhibited apoptosis and decreased TGF‐β1 up‐regulated EMT related protein levels (N‐cadherin and Snail) in vitro via suppression of JNK and Smad3 activation. Moreover, siSmad3 or siJNK impaired TGF‐β1‐induced N‐cadherin and Snail up‐regulation in vitro. Importantly, JNK gene silencing (siERK) also impaired the regulatory effect of TGF‐β1 on Smad3 activation. Our present data demonstrate that RACK1 is a concomitant regulator of TGF‐β1 induces airway apoptosis and EMT via JNK/Smad/Snail signalling axis. Our findings may provide a new insight into understanding the regulation mechanism of RACK1 in asthma pathogenesis.

Cigarette and IL-17A synergistically induce bronchial epithelial-mesenchymal transition via activating IL-17R/NF-\u03baB signaling

BackgroundIL-17A directly induces epithelial-mesenchymal transition (EMT) in alveolar epithelial cells. It could coordinate with cigarette smoke extract (CSE) to promote proliferation of bronchial epithelial cells. In this study, we aim to explore the direct effect of IL-17A and CSE on EMT in bronchial epithelial cells.MethodsBronchial epithelial cells were isolated from C57BL/6 mice, and cocultured with CSE or/and IL-17A. E-cadherin and Vimentin expressions in cells were detected using immunofluorescence staining. IL-17R expression was detected using immunohistochemistry staining. NF-κB expression was assessed using western blotting. When NF-κB was inhibited by BAY 11–7821, expressions of NF-κB, E-cadherin and Vimentin were measured.ResultsThe protein expression of E-cadherin in bronchial epithelial cells was lowest in CSE + IL-17A group, followed by CSE group. In contrast, the protein expression of Vimentin was highest in CSE + IL-17A group, followed by CSE group. Similarly, IL-17R and NF-κB expressions were highest in CSE + IL-17A group, followed by CSE group and IL-17A group. NF-κB inhibitor could inhibit the expressions of E-cadherin and Vimentin.ConclusionsCigarette and IL-17A could synergistically induce EMT in bronchial epithelial cells through activating IL17R/NF-κB signaling. Our findings contribute to a better understanding in airway EMT and pathogenesis of respiratory diseases, which are involved IL-17A and cigarette smoking. Those will provide novel avenues in the immunotherapy of lung diseases.

Hydrogen sulfide attenuates cigarette smoke-induced airway remodeling by upregulating SIRT1 signaling pathway

Airway remodeling is one of the characteristics for chronic obstructive pulmonary disease (COPD). The mechanism underlying airway remodeling is associated with epithelial-mesenchymal transition (EMT) in the small airways of smokers and patients with COPD. Sirtuin 1 (SIRT1) is able to reduce oxidative stress, and to modulate EMT. Here, we investigated the effects and mechanisms of hydrogen sulfide (H2S) on pulmonary EMT in vitro and in vivo. We found that H2S donor NaHS inhibited cigarette smoke (CS)-induced airway remodeling, EMT and collagen deposition in mouse lungs. In human bronchial epithelial 16HBE cells, NaHS treatment also reduced CS extract (CSE)-induced EMT, collagen deposition and oxidative stress. Mechanistically, NaHS upregulated SIRT1 expression, but inhibited activation of TGF-β1/Smad3 signaling in vivo and in vitro. SIRT1 inhibition by a specific inhibitor EX527 significantly attenuated or abolished the ability of NaHS to reverse the CSE-induced oxidative stress. SIRT1 inhibition also abolished the protection of NaHS against CSE-induced EMT. Moreover, SIRT1 activation attenuated CSE-induced EMT by modifying TGF-β1-mediated Smad3 transactivation. In conclusion, H2S prevented CS-induced airway remodeling in mice by reversing oxidative stress and EMT, which was partially ameliorated by SIRT1 activation. These findings suggest that H2S may have therapeutic potential for the prevention and treatment of COPD.

Epithelial-mesenchymal transition of small airway epithelium in patients receiving lung tumor surgery with normal lung function and chronic obstructive pulmonary disease

Objective: To investigate the phenomenon of small airway epithelial-mesenchymal transition (EMT) in patients with normal pulmonary function and chronic obstructive pulmonary disease (COPD) who received surgical treatment for lung tumors. Methods: In this study, 52 patients undergoing surgical treatment for lung tumors admitted to the General Hospital of Ningxia Medical University were included from January 2018 to January 2019. According to the preoperative pulmonary function and smoking history, patients were divided into group A (non-smoking with normal pulmonary function group, 15 patients), group B (smoking with normal pulmonary function group, 21 patients), group C (COPD stable phase group, 16 patients). HE staining was performed to observe the pathological changes in small airway and lung tissue. Immunohistochemistry was used to detect the localization and expression of E-cadherin, α-smooth muscle actin (α-SMA) and Vimentin in small airway. Western blot was used to detect the levels of E-cadherin, α-SMA and Vimentin. Results: (1) There were no significant differences in age, gender, and the ratio of disease composition among the three groups (P>0.05), but forced expiratory volume in one second (FEV(1)) as percentage of predicted value (FEV(1)%pred) and FEV(1)/forced vital capacity (FEV(1)/FVC) in group C were lower than those in groups A and B (all P<0.01), while there was no significant difference between group A and group B (P>0.05); (2) the bronchial wall thickness in group B and group C were higher than that of group A [(32.4±2.4) and (54.6±4.9) vs (22.4±2.2) μm], and group C was significantly higher than group B (P=0.003); (3) the expression levels of E-cadherin in the epithelial cells of small airway in group B and group C were lower than those in group A (0.021±0.008 and 0.018±0.007 vs 0.062±0.010) (all P<0.05), while the levels of mesenchymal cell markers such as α-SMA and Vimentin in group B and group C were higher than group A, and group C was higher than group B (α-SMA: 0.641±0.113, 0.780±0.133 vs 0.404±0.123; Vimentin: 0.064±0.033, 0.083±0.022 vs 0.030±0.021) (P=0.002 and P=0.003). Conclusion: In patients undergoing surgical treatment of lung tumors, there is EMT in the small airways of patients with COPD, and EMT has occurred in the small airways of smokers with normal pulmonary function.

Dual Role of RACK1 in Airway Epithelial Cells: A Concomitant Regulator of TGF-β1 Induced Apoptosis and Epithelial Mesenchymal Transition

Background: Airway epithelial apoptosis and epithelial mesenchymal transition (EMT) are two crucial components of asthma pathogenesis, concomitantly mediated by TGF-β1. RACK1 is a downstream target gene of TGF-β1 shown to enhancement in asthma mice in our previous study. Methods: The Balb/c mice were sensitized with OVA twice and challenged with OVA every day for 7 days. Transformed human bronchial epithelial cells, BEAS-2B cells were cultured and exposure to recombinant soluble human TGF-β1 to induced apoptosis (30 ng/ml, 72 h) and EMT (10 ng/ml, 48 h) in vitro, respectively. siRNA and pharmacological inhibitors were used to evaluate the regulation of RACK1 protein in apoptosis and EMT. Western blotting analysis and immunostaining were used to detect the protein expressions in vivo, and in vitro. Findings: Our data showed that RACK1 protein levels were significantly increased in OVA challenged mice and TGF-β1 induced apoptosis and EMT of BEAS-2B cells. Knockdown of RACK1 (siRACK1) significantly inhibited apoptosis and decreased TGF-β1 up-regulated EMT related protein levels (N-cadherin and Snail) in vitro via suppression of JNK and Smad3 activation. Moreover, siSmad3 or siJNK impaired TGF-β1 induced N-cadherin and Snail up-regulation in vitro. Importantly, JNK gene silencing (siERK) also impaired the regulatory effect of TGF-β1 on Smad3 activation. Interpretation: Our present data demonstrate that RACK1 is a concomitant regulator of TGF-β1 induces airway apoptosis and EMT via JNK/Smad/Snail signaling axis. Therefore, our findings may provide a new insight into understanding the regulation mechanism of RACK1 in asthma pathogenesis. Funding Statement: This study was supported by the National Natural Science Foundation of China (No. 81470224 to Lihong Wan). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Animal ethics approval has been obtained from West China School of Basic Medical Sciences and Forensic Medicine.