Epithelial-Mesenchymal Transition Mechanisms in Chronic Airway Diseases: A Common Process to Target?

Epithelial-to-mesenchymal transition (EMT) is a reversible process, in which epithelial cells lose their epithelial traits and acquire a mesenchymal phenotype. This transformation has been described in different lung diseases, such as lung cancer, interstitial lung diseases, asthma, chronic obstructive pulmonary disease and other muco-obstructive lung diseases, such as cystic fibrosis and non-cystic fibrosis bronchiectasis. The exaggerated chronic inflammation typical of these pulmonary diseases can induce molecular reprogramming with subsequent self-sustaining aberrant and excessive profibrotic tissue repair. Over time this process leads to structural changes with progressive organ dysfunction and lung function impairment. Although having common signalling pathways, specific triggers and regulation mechanisms might be present in each disease. This review aims to describe the various mechanisms associated with fibrotic changes and airway remodelling involved in chronic airway diseases. Having better knowledge of the mechanisms underlying the EMT process may help us to identify specific targets and thus lead to the development of novel therapeutic strategies to prevent or limit the onset of irreversible structural changes.

One-minute sit-to-stand test is practical to assess and follow the muscle weakness in cystic fibrosis.

BACKGROUND: Quadriceps muscle weakness and reduced exercise tolerance are prevalent and associated with a worse prognosis in patients with cystic fibrosis (CF). The one-minute sit-to-stand test (1STST) has been proposed to evaluate functional exercise capacity and quadriceps strength. RESEARCH QUESTION: The aim of the study was to verify the relationship between the 1STST and the maximal isometric voluntary contraction of the quadriceps (MVCQ) evaluated by the dynamometer in stable patients with CF and to evaluate the impact of intravenous (IV) antibiotherapy. METHODS: Dynamometer and 1STST were performed in stable patients with CF at a routine visit, the admission and the discharge of an IV antibiotherapy. Patients wore an activity monitor during 72 h during IV treatment. RESULTS AND SIGNIFICANCE: 51 stable patients with CF at a routine visit and 30 treated with IV antibiotherapy were recruited. In stable patients, the 1STST was reduced to a mean of 2101 nxkg (657-SD), representing a median of 79% (7; 142-min; max)) of the predicted values (%PV) as well as the MVCQ to 78.64 N-m (23.21; 170.34), representing 57%PV (26). The 1STST was correlated to MVCQ (r = 0.536; p < 0.0001) and lung function (r = 0.508; p = 0.0001). Over the IV antibiotherapy course, the 1STST improves significantly like lung function and body mass index while a positive trend for MVCQ was observed. The gain of 1STST was correlated to the change in MVCQ (r = 0.441; p = 0.02) and was significantly higher in hospitalized patients versus home therapy. The 1STST is a good alternative to the dynamometer to evaluate and assess muscular weakness for the routine visit and IV antibiotherapy.

Effects of rehabilitation methods on lower-limb muscle function and functional performance in patients with cystic fibrosis: A systematic review.

OBJECTIVE: To investigate the effects of rehabilitation methods on leg muscle function and functional performance in cystic fibrosis. DATA SOURCES: A literature search was conducted in PubMed (MEDLINE), Scopus and the Cochrane Library from inception to October 12, 2020. A secondary hand search through reference lists from identified articles was conducted. REVIEW METHODS: Three authors independently checked the full-text copies for eligibility of relevant articles. Randomized controlled trials were included. Methodological quality was assessed using the Physiotherapy Evidence Database scale. The PRISMA guidelines were followed. Results suggestive of leg muscle function (e.g. strength, power, endurance, and fatigue) and functional performance were reported. RESULTS: The search identified 8 studies (233 patients). The overall quality of these articles was good. Rehabilitation modalities investigated were physical exercises (aerobic, anaerobic and resistance training) (n = 7) and neuromuscular electrical stimulation (n = 1). Most studies (n = 4) reported on the effects of combined aerobic and resistance training and showed improvement on leg strength when exercises were supervised. Aerobic training alone or the use of neuromuscular electrical stimulation also enhanced leg strength, whereas anaerobic training did not. Two studies evaluated leg fatigue and found no improvement compared to standard care. One study assessed the effect of exercises on functional performance and found no improvement when compared to standard care. No studies emphasized the impact of rehabilitation methods on leg muscle endurance or power. CONCLUSION: Combined aerobic and resistance training enhances leg muscle strength in cystic fibrosis. There is insufficient data on other leg muscle outcomes, nor on alternative rehabilitation strategies.

Key role of the epithelium in chronic upper airways diseases.

The respiratory epithelium of the upper airways is a first-line defence against inhaled irritants, pathogens and allergens. It ensures a physical barrier provided by apical junctions and mucociliary clearance to avoid excessive activation of the immune system. The epithelium also forms a chemical and immunological barrier, extensively equipped to protect the airways against external aggressions before the adaptive immune system is required. Under normal circumstances, the epithelium is capable of recovering rapidly after damage. This manuscript reviews these main properties of the upper airway epithelium as well as its reported impairments in chronic inflammatory diseases. The knowledge on normal epithelial functions and their dysregulation in upper airway diseases should help to design new epithelial-targeted treatments.

Bronchial Epithelial IgA Secretion Is Impaired in Asthma. Role of IL-4/IL-13.

RATIONALE: Asthma is associated with increased lung IgE production, but whether the secretory IgA system is affected in this disease remains unknown. OBJECTIVES: We explored mucosal IgA transport in human asthma and its potential regulation by T-helper cell type 2 inflammation. METHODS: Bronchial biopsies from asthma and control subjects were assayed for bronchial epithelial polymeric immunoglobulin receptor (pIgR) expression and correlated to T-helper cell type 2 biomarkers. Bronchial epithelium reconstituted in vitro from these subjects, on culture in air-liquid interface, was assayed for pIgR expression and regulation by IL-4/IL-13. MEASUREMENTS AND MAIN RESULTS: Downregulation of pIgR protein was observed in the bronchial epithelium from patients with asthma (P = 0.0002 vs. control subjects). This epithelial defect was not observed ex vivo in the cultured epithelium from patients with asthma. Exogenous IL-13 and IL-4 could inhibit pIgR expression and IgA transcytosis. Mechanistic experiments showed that autocrine transforming growth factor-ß mediates the IL-4/IL-13 effect on the pIgR, with a partial contribution of upregulated transforming growth factor-α/epidermal growth factor receptor. CONCLUSIONS: This study shows impaired bronchial epithelial pIgR expression in asthma, presumably affecting secretory IgA-mediated frontline defense as a result of type 2 immune activation of the transforming growth factor pathway.

Airway Epithelium Dysfunction in Cystic Fibrosis and COPD.

Cystic fibrosis is a genetic disease caused by mutations in the CFTR gene, whereas chronic obstructive pulmonary disease (COPD) is mainly caused by environmental factors (mostly cigarette smoking) on a genetically susceptible background. Although the etiology and pathogenesis of these diseases are different, both are associated with progressive airflow obstruction, airway neutrophilic inflammation, and recurrent exacerbations, suggesting common mechanisms. The airway epithelium plays a crucial role in maintaining normal airway functions. Major molecular and morphologic changes occur in the airway epithelium in both CF and COPD, and growing evidence suggests that airway epithelial dysfunction is involved in disease initiation and progression in both diseases. Structural and functional abnormalities in both airway and alveolar epithelium have a relevant impact on alteration of host defences, immune/inflammatory response, and the repair process leading to progressive lung damage and impaired lung function. In this review, we address the evidence for a critical role of dysfunctional airway epithelial cells in chronic airway inflammation and remodelling in CF and COPD, highlighting the common mechanisms involved in the epithelial dysfunction as well as the similarities and differences of the two diseases.

Imprinting of the COPD airway epithelium for dedifferentiation and mesenchymal transition.

In chronic obstructive pulmonary disease (COPD), epithelial changes and subepithelial fibrosis are salient features in conducting airways. Epithelial-to-mesenchymal transition (EMT) has been recently suggested in COPD, but the mechanisms and relationship to peribronchial fibrosis remain unclear. We hypothesised that de-differentiation of the COPD respiratory epithelium through EMT could participate in airway fibrosis and thereby, in airway obstruction. Surgical lung tissue and primary broncho-epithelial cultures (in air-liquid interface (ALI)) from 104 patients were assessed for EMT markers. Cell cultures were also assayed for mesenchymal features and for the role of transforming growth factor (TGF)-ß1. The bronchial epithelium from COPD patients showed increased vimentin and decreased ZO-1 and E-cadherin expression. Increased vimentin expression correlated with basement membrane thickening and airflow limitation. ALI broncho-epithelial cells from COPD patients also displayed EMT phenotype in up to 2 weeks of culture, were more spindle shaped and released more fibronectin. Targeting TGF-ß1 during ALI differentiation prevented vimentin induction and fibronectin release. In COPD, the airway epithelium displays features of de-differentiation towards mesenchymal cells, which correlate with peribronchial fibrosis and airflow limitation, and which are partly due to a TGF-ß1-driven epithelial reprogramming.

Polymeric immunoglobulin receptor down-regulation in chronic obstructive pulmonary disease. Persistence in the cultured epithelium and role of transforming growth factor-ß.

RATIONALE: The generation of protective secretory IgA relies on the epithelial polymeric immunoglobulin receptor (pIgR). pIgR expression is reduced in chronic obstructive pulmonary disease (COPD), but correlation to disease severity and underlying mechanisms remains unknown. OBJECTIVES: To address the hypothesis that pIgR down-regulation in COPD concerns severe disease in relation to aberrant programming of the bronchial epithelium. METHODS: Surgical lung tissue and primary bronchial epithelium (cultured in air-liquid interface, ALI) obtained from a large series of patients (n = 116) were studied for pIgR expression and regulation. MEASUREMENTS AND MAIN RESULTS: pIgR immunostaining in the bronchial epithelium is decreased in severe COPD. In contrast, pIgR transcription was up-regulated in smokers with or without COPD. In ALI (vs. submerged) cultures, pIgR expression was strongly induced, whereas pIgR expression and IgA-transcytosis capacity were decreased in cultures from subjects with severe COPD as compared with control subjects. In addition, COPD cultures released more transforming growth factor-ß1 (TGF-ß1), reflecting increased epithelial TGF-ß1 immunostaining in COPD lung tissue. Finally, besides inducing epithelial dedifferentiation, exogenous TGF-ß1 dose-dependently inhibited pIgR production, whereas pIgR increased on blockade of TGF-ß1 activity during ALI differentiation. CONCLUSIONS: pIgR down-regulation in COPD correlates with disease severity, and the bronchial epithelium reconstituted in vitro from these patients retains its aberrant imprinting for pIgR expression. This study also links pIgR down-regulation to TGF-ß-driven reprogramming of the bronchial epithelium, which results in impaired lung IgA immunity in patients with COPD.

Novel time-saving OGTT sparing HbA1c-HOMA2 based algorithm for the diagnosis of cystic fibrosis-related diabetes.

AIMS: The diagnosis of cystic fibrosis-related diabetes (CFRD) faces several challenges. We propose a novel screening algorithm to alleviate the burden of cystic fibrosis (CF). METHODS: Through a retrospective cross-sectional single-centre study, HbA1c and HOMA2 indices were assessed in multiple models as alternative diagnostic tools from OGTT data. We sought to establish specific thresholds for CFRD screening with oral glucose tolerance test (OGTT) as gold standard. We evaluated various straightforward or sequential approaches, in terms of diagnostic accuracy while also quantify the potential reduction in OGTTs through these different methods. RESULTS: HOMA indices were recovered in 72 patients. We devised a composite index that combines HbA1c and HOMA-B: Diabetes Predicting Index in cystic fibrosis (DIPIc) = (HbA1c(%) × 3.455) - (HOMA-B(%) ×  0.020) - 19.294. This index yields the highest screening accuracy according to receiver-operating characteristics curves. Using a stepwise algorithm that incorporates DIPIc decreases the requirement for annual OGTTs. A CFRD exclusion cutoff less than -1.7445 (sensitivity 98 %), in conjunction with a CFRD diagnostic threshold greater than 0.4543 (specificity 98 %) allows for 71 % OGTT sparing. CONCLUSION: The composite index DIPIc is a suitable, less invasive screening method for CFRD, which enables to avoid many OGTTs.

The intermittent intrapulmonary deflation technique for airway clearance in patients with cystic fibrosis: A randomized trial.

BACKGROUND: Cystic fibrosis (CF) is a muco-obstructive lung disease characterized by thick sputum with abnormal rheological properties. The intermittent intrapulmonary deflation (IID) is a new instrumental airway clearance technique (ACT) that aims to decrease the sputum viscoelastic properties. This study assessed the benefits of adding the IID technique to a conventional ACT in patients with CF hospitalized for intravenous antibiotic therapy. METHODS: Participants with CF accustomed to autogenic drainage (AD) as their standard ACT received, in a randomized order, a 30-min session of either AD alone or AD combined with IID (AD+IID). Sputum was collected during each ACT regimens and for a 24-hour period following both sessions. Sputum wet weight, dry weight, solids content and rheological properties were analyzed. Cough events occurring during and over 2 h post ACT were compared between both regimens. RESULTS: Seventeen patients with CF (aged 29 ± 11 years; FEV1%: 57.1 ± 20.1) were analysed. The sputum wet weight collected during AD alone was significantly higher than during AD+IID (8.11 ± 6.93 vs 5.40 ± 4.11 respectively, p = 0.01). The sputum rheological properties did not significantly differ between group. There were more cough episodes during AD alone compared to AD+IID (median [IQR]: 8 [5-15.5] vs 5 [3.5-11.0] respectively, p = 0.02). CONCLUSIONS: In participants with CF accustomed to AD, adding the IID technique in combination to AD does not confer a clear benefit on airway clearance in the short term. Clinical Trials register: NCT04157972.

One year effect of tezacaftor and ivacaftor on functional exercise capacity and muscle strength in people with cystic fibrosis.

Background: The 1-min sit-to-stand test (1STST) is a practical tool to evaluate physical capacity. The aim of this study was to assess the impact of tezacaftor and ivacaftor on functional exercise capacity, muscle strength and symptoms in people with cystic fibrosis (PwCF). Methods: The assessments were performed during the first year of tezacaftor and ivacaftor using the 1STST, 6-min walk test (6MWT), MicroFET2 dynamometer®, CF Questionnaire-Revised (CFQ-R), Leicester Cough Questionnaire (LCQ). Forced expiratory volume in 1 s (FEV1), body mass index (BMI), pancreatic sufficiency status, genotype and microbiologic data were also collected. Results: Fifty-four PwCF participated to the study and took at least one dose of tezacaftor-ivacaftor. Mean age was 26y±10 (±SD), median BMI 20.9 kg/m2 (interquartile range) (19.4; 23.5) and mean FEV1 82 percent of predicted values (%PV) ± 21. Significant correlations were found at baseline between the 1STST and the 6MWT (r = 0.617, p < 0.0001), the quadriceps strength (r = 0.6556, p < 0.0001) and the FEV1 (r = 0.29, p = 0.03). After one year of treatment, the 1STST increased significantly in terms of number of repetitions (n) (median 50 versus 58.5, p < 0.0001), %PV (101.1 versus 115.2%PV, p = 0.0003) and n times weight in kg (2885 versus 3389nxkg, p < 0.0001). The 6MWT distance and quadriceps strength were not modified after treatment but during the 6MWT, oxygen desaturation decreased significantly. FEV1, BMI, CFQ-R, LCQ improved as previously demonstrated. Conclusion: After one year of tezacaftor and ivacaftor, the 1STST improves, suggesting that the 1STST seems more responsive than the 6MWT and the MicroFET2 dynamometer® to assess the effects of CFTR modulators.

The memory of airway epithelium damage in smokers and COPD patients.

Chronic obstructive pulmonary disease (COPD), a devastating and irreversible lung disease, causes structural and functional defects in the bronchial epithelium, the (ir)reversibility of which remains unexplored in vitro. This study aimed to investigate the persistence of COPD-related epithelial defects in long-term airway epithelial cultures derived from non-smokers, smokers, and COPD patients. Barrier function, polarity, cell commitment, epithelial-to-mesenchymal transition, and inflammation were evaluated and compared with native epithelium characteristics. The role of inflammation was explored using cytokines. We show that barrier dysfunction, compromised polarity, and lineage abnormalities observed in smokers and COPD persisted for up to 10 wk. Goblet cell hyperplasia was associated with recent cigarette smoke exposure. Conversely, increased IL-8/CXCL-8 release and abnormal epithelial-to-mesenchymal transition diminished over time. These ex vivo observations matched surgical samples' abnormalities. Cytokine treatment induced COPD-like changes in control cultures and reactivated epithelial-to-mesenchymal transition in COPD cells. In conclusion, these findings suggest that the airway epithelium of smokers and COPD patients retains a multidimensional memory of its original state and previous cigarette smoke-induced injuries, maintaining these abnormalities for extended periods.

Combined CFTR modulator therapies are linked with anabolic benefits and insulin-sparing in cystic fibrosis-related diabetes.

Aims: Combined CFTR modulator therapies have dramatically altered pulmonary outcomes in patients with cystic fibrosis (CF). Their impact on glucose metabolism requires further investigations. This study aims to evaluate insulin requirements after initiation of combined CFTR modulator therapy in patients with CF-related diabetes (CFRD) and HOMA indices changes in CF patients without diabetes. Methods: We retrospectively analyzed: 1) the effects of tezacaftor + ivacaftor and elexacaftor + tezacaftor + ivacaftor on FEV1, weight, BMI, HbA1c, and daily insulin dose, in 17 CFRD patients and 2) the impact of tezacaftor + ivacaftor on HOMA indices in 15 CF patients without diabetes. Results: Age was 37±12y in the CFRD group (70% men), 88% of whom were homozygous for F508del mutation. Diabetes duration was 15±10y. Median duration of combined CFTR modulator therapy was 16 months (IQR: 4) Thirteen patients received tezacaftor + ivacaftor, of whom 9 were switched to elexacaftor + tezacaftor + ivacaftor. Four patients received elexacaftor + tezacaftor + ivacaftor up front. A decrease in insulin needs was noticed in 88% of patients (0.85±0.3 vs 0.71±0.3U/kg/day; p = 0001). Total daily insulin dose decreased from 50±16 to 44±20U/day (p = 0.017). BMI improved (20.9 (IQR: 1.90) vs 22.1 kg/m2 (IQR: 3.70); p = 0.014). HbA1c went from 7.3±1.1 to 7.7±1.6% (p = 0.072). Median age was 22y (IQR: 11) in the CF group without diabetes (67% men), 93% of whom were homozygous for F508del mutation. Duration of combined CFTR modulator therapy was 10±5 months. HOMA-B changes were not significant (129.2 (IQR: 84.8) vs 103.5% (IQR: 66.3) nor were HOMA-S changes (from 94±64 to 95±49%). HOMA-BxS decreased from 112±45 to 104±29% (NS). BMI rose from 21.9±3 to 23.1±3.5 kg/m2 (p = 0.047). HbA1c was unchanged (5.0±0.5%). FEV1 improved in both groups (+11% and + 7% of predicted value; p < 0.001; p = 0.013). Conclusion: Combined CFTR modulator therapies are correlated with a decrease in insulin doses and positive effects on BMI and FEV1. HOMA indices did not change on tezacaftor + ivacaftor among CF patients without diabetes.

Comparison of amikacin lung delivery between AKITA® and eFlow rapid® nebulizers in healthy controls and patients with CF: A randomized cross-over trial.

INTRODUCTION: Nebulization plays a key role in the treatment of cystic fibrosis. The Favorite function couple to jet nebulizers (AKITA®) emerged recently. The aim of this study was to assess the efficiency of the lung delivery by the AKITA® by comparing the urinary concentration of amikacin after nebulization with the AKITA® and the eFlow rapid®, in healthy subjects and patients with CF (PwCF). METHOD: The two samples (healthy subjects and PwCF) were randomized (cross-over 1:1) for two nebulizations (500 mg of amikacin diluted in 4 mL of normal saline solution), with the AKITA® and with the eFlow rapid®. The primary endpoint was the amount of urinary excretion of amikacin over 24 h. The constant of elimination (Ke) was calculated based on the maximal cumulative urinary amikacin excretion plotted over time. RESULTS: The total amount of urinary amikacin excretion was greater when AKITA® was used in PwCF (11.7 mg (8.2-14.1) vs 6.1 mg (3.7-13.3); p = 0.02) but not different in healthy subjects (14.5 mg (11.7-18.5) vs 12.4 mg (8.0-17.1); p = 0.12). The duration of the nebulization was always shorter with eFlow rapid® than with AKITA® (PwCF: 6.5 ± 0.6 min vs 9.2 ± 1.8 min; p = 0.001 - Healthy: 4.7 ± 1.3 min vs 9.7 ± 1.6 min; p = 0.03). The constant of elimination was similar between the two modalities in CF subjects (0.153 (0.071-0.205) vs 0.149 (0.041-0.182); p = 0.26) and in healthy subjects (0.166 (0.130-0.218) vs 0.167 (0.119-0.210), p = 0.25). CONCLUSION: the Favorite inhalation is better to deliver a specific amount of drug than a mesh nebulizer (eFlow rapid®) in PwCF but not in healthy subjects.

Mucosal immune alterations at the early onset of tissue destruction in chronic obstructive pulmonary disease.

Rationale: COPD is characterized by chronic airway inflammation, small airways changes, with disappearance and obstruction, and also distal/alveolar destruction (emphysema). The chronology by which these three features evolve with altered mucosal immunity remains elusive. This study assessed the mucosal immune defense in human control and end-stage COPD lungs, by detailed microCT and RNA transcriptomic analysis of diversely affected zones. Methods: In 11 control (non-used donors) and 11 COPD (end-stage) explant frozen lungs, 4 cylinders/cores were processed per lung for microCT and tissue transcriptomics. MicroCT was used to quantify tissue percentage and alveolar surface density to classify the COPD cores in mild, moderate and severe alveolar destruction groups, as well as to quantify terminal bronchioles in each group. Transcriptomics of each core assessed fold changes in innate and adaptive cells and pathway enrichment score between control and COPD cores. Immunostainings of immune cells were performed for validation. Results: In mildly affected zones, decreased defensins and increased mucus production were observed, along CD8+ T cell accumulation and activation of the IgA pathway. In more severely affected zones, CD68+ myeloid antigen-presenting cells, CD4+ T cells and B cells, as well as MHCII and IgA pathway genes were upregulated. In contrast, terminal bronchioles were decreased in all COPD cores. Conclusion: Spatial investigation of end-stage COPD lungs show that mucosal defense dysregulation with decreased defensins and increased mucus and IgA responses, start concomitantly with CD8+ T-cell accumulation in mild emphysema zones, where terminal bronchioles are already decreased. In contrast, adaptive Th and B cell activation is observed in areas with more advanced tissue destruction. This study suggests that in COPD innate immune alterations occur early in the tissue destruction process, which affects both the alveoli and the terminal bronchioles, before the onset of an adaptive immune response.

Secretory Immunoglobulin A Immunity in Chronic Obstructive Respiratory Diseases.

Chronic obstructive pulmonary disease (COPD), asthma and cystic fibrosis (CF) are distinct respiratory diseases that share features such as the obstruction of small airways and disease flare-ups that are called exacerbations and are often caused by infections. Along the airway epithelium, immunoglobulin (Ig) A contributes to first line mucosal protection against inhaled particles and pathogens. Dimeric IgA produced by mucosal plasma cells is transported towards the apical pole of airway epithelial cells by the polymeric Ig receptor (pIgR), where it is released as secretory IgA. Secretory IgA mediates immune exclusion and promotes the clearance of pathogens from the airway surface by inhibiting their adherence to the epithelium. In this review, we summarize the current knowledge regarding alterations of the IgA/pIgR system observed in those major obstructive airway diseases and discuss their implication for disease pathogenesis.