Epithelial to mesenchymal transition in murine tracheal allotransplantation: an immunohistochemical observation.
ABSTRACT: Aberrant epithelial repair is a crucial event in the airway remodeling that characterizes obliterative bronchiolitis (OB) in transplanted lungs. Recent data from experiments using epithelial cell lines and human airway tissues from lung transplant recipients suggest that epithelial to mesenchymal transition (EMT) plays an important role in OB. The aim of this study was to clarify whether EMT is involved in airway remodeling in an animal model.We performed orthotopic tracheal transplantation from BALB/c to C57BL/6 mice with from BALC/c to BALB/c mouse grafts as controls. Five allogeneic and 3 syngeneic recipients were humanely killed at predetermined postoperative days 2-12 as well as 14 and 21. Histology was evaluated using hematoxylin-eosin (H&E) staining. We studied the expression of specific markers, including E-cadherin, an epithelial marker; ?-smooth muscle actin (SMA), and S100A4, mesenchymal markers, and zinc finger E-box-binding homeobox 1 (ZEB1), an EMT-related transcription factor.Histologic assessment of serial H&E stains of allogeneic grafts showed remarkable pseudostratified respiratory epithelium with subepithelial inflammatory cell infiltration, as well as denuded and flattened epithelium and subepithelial fibrosis. The dynamic epithelial changes occurred earlier than the subepithelial fibrosis. Immunohistochemical evaluation indicated the emergence of ?-SMA- positive epithelial cells that were most prominent on day 7. The expression of E-cadherin was attenuated in ?-SMA-positive epithelial cells. S100A4 was also expressed in epithelial cells. A few days before the intraepithelial expression of ?-SMA, ZEB1 emerged in the nuclei of epithelial cells.We observed expression of an EMT-related transcription factor and mesenchymal markers along with the attenuation of epithelial marker expression in epithelial cells, several days before prominent subepithelial fibrosis formation, results that suggest epithelial cells to play an important fibrosis role in airway remodeling during epithelial to mesenchymal transition.
Project description:Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-?1) levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (?-SMA) and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease.
Project description:Repetitive aeroallergen exposure is linked to sensitization and airway remodeling through incompletely understood mechanisms. In this study, we examine the dynamic mucosal response to cat dander extract (CDE), a ubiquitous aero-allergen linked to remodeling, sensitization and asthma. We find that daily exposure of CDE in naïve C57BL/6 mice activates innate neutrophilic inflammation followed by transition to a lymphocytic response associated with waves of mucosal transforming growth factor (TGF) isoform expression. In parallel, enhanced bronchiolar Smad3 expression and accumulation of phospho-SMAD3 was observed, indicating paracrine activation of canonical TGFβR signaling. CDE exposure similarly triggered epithelial cell plasticity, associated with expression of mesenchymal regulatory factors (Snai1 and Zeb1), reduction of epithelial markers (Cdh1) and activation of the NFκB/RelA transcriptional activator. To determine whether NFκB functionally mediates CDE-induced growth factor response, mice were stimulated with CDE in the absence or presence of a selective IKK inhibitor. IKK inhibition substantially reduced the level of CDE-induced TGFβ1 expression, pSMAD3 accumulation, Snai1 and Zeb1 expression. Activation of epithelial plasticity was demonstrated by flow cytometry in whole lung homogenates, where CDE induces accumulation of SMA<sup>+</sup>Epcam<sup>+</sup> population. Club cells are important sources of cytokine and growth factor production. To determine whether Club cell innate signaling through NFκB/RelA mediated CDE induced TGFβ signaling, we depleted RelA in Secretoglobin (Scgb1a1)-expressing bronchiolar cells. Immunofluorescence-optical clearing light sheet microscopy showed a punctate distribution of Scgb1a1 progenitors throughout the small airway. We found that RelA depletion in Secretoglobin<sup>+</sup> cells results in inhibition of the mucosal TGFβ response, blockade of EMT and reduced subepithelial myofibroblast expansion. We conclude that the Secretoglobin-derived bronchiolar cell is central to coordinating the innate response required for mucosal TGFβ1 response, EMT and myofibroblast expansion. These data have important mechanistic implications for how aero-allergens trigger mucosal injury response and remodeling in the small airway.
Project description:BACKGROUND: Epithelial to mesenchymal transition (EMT) in alveolar epithelial cells (AECs) has been widely observed in patients suffering interstitial pulmonary fibrosis. In vitro studies have also demonstrated that AECs could convert into myofibroblasts following exposure to TGF-beta1. In this study, we examined whether EMT occurs in bleomycin (BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells (BECs) in the EMT. Using an alpha-smooth muscle actin-Cre transgenic mouse (alpha-SMA-Cre/R26R) strain, we labelled myofibroblasts in vivo. We also performed a phenotypic analysis of human BEC lines during TGF-beta1 stimulation in vitro. METHODS: We generated the alpha-SMA-Cre mouse strain by pronuclear microinjection with a Cre recombinase cDNA driven by the mouse alpha-smooth muscle actin (alpha-SMA) promoter. alpha-SMA-Cre mice were crossed with the Cre-dependent LacZ expressing strain R26R to produce the double transgenic strain alpha-SMA-Cre/R26R. beta-galactosidase (betagal) staining, alpha-SMA and smooth muscle myosin heavy chains immunostaining were carried out simultaneously to confirm the specificity of expression of the transgenic reporter within smooth muscle cells (SMCs) under physiological conditions. BLM-induced peribronchial fibrosis in alpha-SMA-Cre/R26R mice was examined by pulmonary betagal staining and alpha-SMA immunofluorescence staining. To confirm in vivo observations of BECs undergoing EMT, we stimulated human BEC line 16HBE with TGF-beta1 and examined the localization of the myofibroblast markers alpha-SMA and F-actin, and the epithelial marker E-cadherin by immunofluorescence. RESULTS: betagal staining in organs of healthy alpha-SMA-Cre/R26R mice corresponded with the distribution of SMCs, as confirmed by alpha-SMA and SM-MHC immunostaining. BLM-treated mice showed significantly enhanced betagal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some AECs in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by alpha-SMA immunostaining. In vitro, addition of TGF-beta1 to 16HBE cells could also stimulate the expression of alpha-SMA and F-actin, while E-cadherin was decreased, consistent with an EMT. CONCLUSION: We observed airway EMT in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.
Project description:Chronic allergic asthma leads to airway remodeling and subepithelial fibrosis via mechanisms not fully understood. Airway remodeling is amplified by profibrotic mediators, such as transforming growth factor-?1 (TGF-?1), which plays a cardinal role in various models of fibrosis. We recently have identified a critical role for c-Jun-NH2-terminal-kinase (JNK) 1 in augmenting the profibrotic effects of TGF-?1, linked to epithelial-to-mesenchymal transition of airway epithelial cells. To examine the role of JNK1 in house dust mite (HDM)-induced airway remodeling, we induced allergic airway inflammation in wild-type (WT) and JNK1-/- mice by intranasal administration of HDM extract. WT and JNK1-/- mice were sensitized with intranasal aspirations of HDM extract for 15 days over 3 wk. HDM caused similar increases in airway hyperresponsiveness, mucus metaplasia, and airway inflammation in WT and JNK1-/- mice. In addition, the profibrotic cytokine TGF-?1 and phosphorylation of Smad3 were equally increased in WT and JNK1-/- mice. In contrast, increases in collagen content in lung tissue induced by HDM were significantly attenuated in JNK1-/- mice compared with WT controls. Furthermore HDM-induced increases of ?-smooth muscle actin (?-SMA) protein and mRNA expression as well as the mesenchymal markers high-mobility group AT-hook 2 and collagen1A1 in WT mice were attenuated in JNK1-/- mice. The let-7 family of microRNAs has previously been linked to fibrosis. HDM exposure in WT mice and primary lung epithelial cells resulted in striking decreases in let-7g miRNA that were not observed in mice or primary lung epithelial cells lacking JNK1-/- mice. Overexpression of let-7g in lung epithelial cells reversed the HDM-induced increases in ?-SMA. Collectively, these findings demonstrate an important requirement for JNK1 in promoting HDM-induced fibrotic airway remodeling.
Project description:Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-?1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-?1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-?1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.
Project description:Chronic epithelial injury triggers a TGF-?-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-? induces the mesenchymal cell state and determined its mechanism. We observed that TGF-? stimulation activates an inflammatory gene program controlled by the NF-?B/RelA signaling pathway. In the mesenchymal state, NF-?B-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-?B/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-? challenge in a mouse model. These data provide a detailed mechanism for how activated NF-?B and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases.
Project description:BACKGROUND: Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling. METHODS: Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters. RESULTS: A median 15% (0-48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-beta1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-beta1 stimulation. CONCLUSIONS: This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.
Project description:<h4>Background</h4>S100 proteins have been implicated in various aspects of cancer, including epithelial-mesenchymal transitions (EMT), invasion and metastasis, and also in inflammatory disorders. Here we examined the impact of individual members of this family on the invasion of pancreatic ductal adenocarcinoma (PDAC) cells, and their regulation by EMT and inflammation.<h4>Methods</h4>Invasion of PDAC cells was analysed in zebrafish embryo xenografts and in transwell invasion assays. Expression and regulation of S100 proteins was studied in vitro by immunoblotting, quantitative PCR and immunofluorescence, and in pancreatic lesions by immunohistochemistry.<h4>Results</h4>Whereas the expression of most S100 proteins is characteristic for epithelial PDAC cell lines, S100A4 and S100A6 are strongly expressed in mesenchymal cells and upregulated by ZEB1. S100A4/A6 and epithelial protein S100A14 respectively promote and represses cell invasion. IL-6/11-STAT3 pathway stimulates expression of most S100 proteins. ZEB1 synergises with IL-6/11-STAT3 to upregulate S100A4/A6, but nullifies the effect of inflammation on S100A14 expression.<h4>Conclusion</h4>EMT/ZEB1 and IL-6/11-STAT3 signalling act independently and congregate to establish the expression pattern of S100 proteins, which drives invasion. Although ZEB1 regulates expression of S100 family members, these effects are masked by IL-6/11-STAT3 signalling, and S100 proteins cannot be considered as bona fide EMT markers in PDAC.
Project description:Idiopathic pulmonary fibrosis (IPF) is a progressive and devastating interstitial lung disease. The origin of myofibroblasts is still to be elucidated and the existence of epithelial-mesenchymal transition (EMT) in IPF remains controversial. Hence, it is important to clarify the origin of fibroblasts by improving modeling and labeling methods and analyzing the differentiation pathway of alveolar epithelial cells (AEC) in pulmonary fibrosis with cell tracking technology. In the present study, adult transgenic mice with <i>SPC-rtTA</i> <sup>+/-</sup> <i>/tetO</i> <sub>7</sub> <i>-CMV-Cre</i> <sup>+/-</sup> <i>/mTmG</i> <sup>+/-</sup> were induced with doxycycline for 15 days. The gene knockout phenomenon occurred in type II AECs in the lung and the reporter gene cell membrane-localized enhanced green fluorescence protein (mEGFP) was expressed in the cell membrane. The expression of Cre recombinase and SPC was analyzed using immunohistochemical (IHC) staining to detect the labeling efficiency. A repetitive intraperitoneal bleomycin-induced pulmonary fibrosis model was established, and the mice were sacrificed on day 28. The co-localization of mEGFP and mesenchymal markers α-smooth muscle actin (α-SMA) and S100 calcium binding protein A4 (S100A4) were detected by multiple IHC staining. The results revealed that Cre was expressed in the airway and AECs in the lung tissue of adult transgenic mice with <i>SPC-rtTA</i> <sup>+/-</sup> <i>/tetO</i> <sub>7</sub> <i>-CMV-Cre</i> <sup>+/-</sup> <i>/mTmG</i> <sup>+/-</sup> induced by doxycycline; the labeling efficiency in the peripheral lung tissue was 63.27±7.51%. mEGFP was expressed on the membrane of type II AECs and their differentiated form of type I AECs. Expression of mEGFP was mainly observed in the fibrotic region in bleomycin-induced pulmonary fibrosis; 1.94±0.08% of α-SMA-positive cells were mEGFP-positive and 9.68±2.06% of S100A4-positive cells were mEGFP-positive in bleomycin-induced pulmonary fibrosis. In conclusion, the present results suggested that while EMT contributes to the pathogenesis of pulmonary fibrosis, it is not the major causative factor of this condition.
Project description:Whether or not cholangiocytes or their hepatic progenitors undergo an epithelial-to-mesenchymal transition (EMT) to become matrix-producing myofibroblasts during biliary fibrosis is a significant ongoing controversy. To assess whether EMT is active during biliary fibrosis, we used Alfp-Cre × Rosa26-YFP mice, in which the epithelial cells of the liver (hepatocytes, cholangiocytes, and their bipotential progenitors) are heritably labeled at high efficiency with yellow fluorescent protein (YFP). Primary cholangiocytes isolated from our reporter strain were able to undergo EMT in vitro when treated with transforming growth factor-?1 alone or in combination with tumor necrosis factor-?, as indicated by adoption of fibroblastoid morphology, intracellular relocalization of E-cadherin, and expression of ?-smooth muscle actin (?-SMA). To determine whether EMT occurs in vivo, we induced liver fibrosis in Alfp-Cre × Rosa26-YFP mice using the bile duct ligation (BDL) (2, 4, and 8 weeks), carbon tetrachloride (CCl(4) ) (3 weeks), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; 2 and 3 weeks) models. In no case did we find evidence of colocalization of YFP with the mesenchymal markers S100A4, vimentin, ?-SMA, or procollagen 1?2, although these proteins were abundant in the peribiliary regions.Hepatocytes and cholangiocytes do not undergo EMT in murine models of hepatic fibrosis.