Increasing evidence suggests that acquired dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel occurs as a result of cigarette smoke exposure in Chronic obstructive pulmonary disease (COPD). CFTR-targeted therapies were developed for the treatment of genetic CFTR defects in cystic fibrosis, but they have not demonstrated consistent clinical efficacy in small trials of patients with COPD. Here, we aimed to characterise CFTR activity, other ion channels, and differentiation to cilia in primary bronchial epithelial cells (pBECs) from COPD donors, compare to healthy controls, and to determine the extent to which CFTR dysfunction can be rescued by clinically relevant CFTR modulators. Conditionally reprogrammed (CR) air-liquid interface (ALI) cultures of pBECs from healthy controls (n = 7) and COPD donors (n = 7) were comprehensively assessed for transepithelial electrical resistance, immunofluorescence, cilia activity, ion channel function and expression of cell markers at transcript level. COPD cultures exhibited reduced active area of ciliated cells, accompanied by significantly decreased forskolin/IBMX stimulated CFTR-mediated Cl- transport and ATP induced calcium activated chloride currents compared to healthy controls cultures, despite preserved CFTR mRNA expression. Treatment with CFTR potentiators (VX-770, GLPG1837 and Icenticaftor) resulted in modest and highly variable functional responses, with no statistically significant improvement compared with vehicle-treated control. Notably, CFTR function did not correlate with cumulative smoking exposure (pack-years), indicating that smoking burden alone does not predict the extent of acquired CFTR dysfunction. Collectively, these findings demonstrate that COPD airway epithelium exhibits intrinsic defects in ion transport and mucociliary differentiation consistent with acquired CFTR dysfunction phenotype but this is not readily reversible with the tested CFTR modulators. These results highlight fundamental differences between genetic and acquired CFTR dysfunction and underscore the need for alternative or combinatorial therapeutic strategies targeting epithelial dysfunction in COPD.

BACKGROUND:People with severe steroid-resistant asthma are refractory to treatment with the mainstay inhaled corticosteroids (ICS), emphasising the urgent need for alternative therapies. This study aimed to assess the effect of endoplasmic reticulum stress (ERS) on steroid responsiveness and to evaluate the efficacy of 4-phenylebuteric acid (4-PBA) as an add-on treatment to restore steroid responses in severe asthma. METHODS:The relationship between ERS and steroid response was assessed by treating human airway epithelial cells (AEC) with chemical ERS inducers or TNF, IFN-γ, and IL-17, and assessing the effects of dexamethasone (Dex) and 4-PBA. The correlation between genes associated with ERS and GR-signalling was assessed in sputum cells from patients with severe asthma, and the effects of 4-PBA were assessed in two murine models of severe, steroid-resistant asthma. RESULTS:Chemical ERS inducers significantly downregulated the expression of corticosteroid-responsive genes, HSD11B2 and FKBP5 and reduced GR nuclear translocation in basal AECs. Treatment with TNF, IFN-γ and IL-17 upregulated ERS and protein misfolded markers while reducing Dex-induced GR nuclear translocation. In sputum cells from patients with severe asthma, ERS genes negatively correlated with GR-signalling. In differentiated primary bronchial epithelial cells (pBECs), treatment with 4-PBA reversed TNF, IFN-γ and IL-17-induced steroid resistance by upregulating HSD11B2 and FKBP5 gene expression and downregulating inflammatory genes. 4-PBA together with Dex significantly reduced airway inflammation and/or AHR in experimental models of severe, steroid-resistant asthma. CONCLUSION:We provide evidence for ERS inducing steroid resistance that underpins severe asthma and demonstrate a therapeutic potential for restoring steroid sensitivity in severe asthma with 4-PBA.

Introduction:Asthma and chronic obstructive pulmonary disease (COPD) overlap (ACO) is a term used to describe a patient with coexisting clinical features of asthma and COPD. We have previously reported that epithelial to mesenchymal transition (EMT) is active in the lungs of patients with COPD however, EMT in ACO remains an unexplored area. We hypothesize that EMT is an active process in ACO. Methods:In this cross-sectional study, large airway endobronchial biopsy (EBB) tissues from patients with asthma (14), COPD (22), current (CS) and ex-smokers (ES), and ACO (12) were immunohistochemically stained for EMT markers (E and N cadherin, vimentin, S100A4, and Collagen IV) and compared with 12 current smokers with normal lung function (NLFS) and 10 non-smoking healthy control (HC) subjects. In addition, air-liquid interface (ALI) cell cultures were performed and cells from patients with ACO and HC were treated with TGF-β, IL-13 and cigarette smoke extract (CSE). Later cells from ALI cultures were lysed for Immunoblotting. Immunostained tissues were enumerated for percent expression of E and N-Cadherin in the epithelium, vimentin and S100A4 positive cells both in the epithelium and reticular basement membrane (RBM). Additionally, the degree of RBM fragmentation was evaluated, a key tissue structural marker of EMT. Results:Compared to healthy controls and asthmatics, ACO had the greatest fragmentation of RBM (P < 0.01). ACO also had substantially decreased percentage expression of E-cadherin (P <0.01), increase percentage of N-cadherin expression, and higher vimentin and S100A4 positive basal cells, in comparison to healthy controls. In the RBM of ACO, S100A4 positive cells (P <0.05) and Vimentin-positive cells were markedly higher in comparison to HC. Similar changes were observed with western blots in response to Th-2 cytokine IL-13, CSE and EMT activator TGF-β. Conclusions:These data are suggestive of active EMT in ACO. Additionally, 50% of the patients with ACO were on 800 mcg/day inhaled corticosteroid (ICS) treatment which may have abrogated some EMT activity; however, it suggests protective effects of ICS as we previously reported in COPD. Studies with larger cohorts are needed to further confirm ICS effects in ACO.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma.NEW & NOTEWORTHY TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion.

Large airway wall lamina propria in patients with asthma-COPD overlap is hypovascular with an increase in reticular basement membrane neoangiogenesis, reflecting smoking-related COPD-like pathology and potential epithelial-to-mesenchymal transition https://bit.ly/49DeoFX.

Background and Objective: Severe asthma is a heterogeneous disease with subtype classification according to dominant airway infiltrates, including eosinophilic (Type 2 high), or non-eosinophilic asthma. Non-eosinophilic asthma is further divided into paucigranulocytic or neutrophilic asthma characterized by elevated neutrophils, and mixed Type 1 and Type 17 cytokines in the airways. Severe non-eosinophilic asthma has few effective treatments and many patients do not qualify for biologic therapies. The cystic fibrosis transmembrane conductance regulator (CFTR) is dysregulated in multiple respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease and has proven a valuable therapeutic target. We hypothesized that the CFTR may also play a role in non-eosinophilic asthma. Methods: Patient-derived human bronchial epithelial cells (hBECs) were isolated and differentiated at the air-liquid interface. Single cell RNA-sequencing (scRNA-seq) was used to identify epithelial cell subtypes and transcriptional activity. Ion transport was investigated with Ussing chambers and immunofluorescent quantification of ionocyte abundance in human airway epithelial cells and murine models of asthma. Results: We identified that hBECs from patients with non-eosinophilic asthma had reduced CFTR function, and did not differentiate into CFTR-expressing ionocytes compared to those from eosinophilic asthma or healthy donors. Similarly, ionocytes were also diminished in the airways of a murine model of neutrophilic-dominant but not eosinophilic asthma. Treatment of hBECs from healthy donors with a neutrophilic asthma-like inflammatory cytokine mixture led to a reduction in ionocytes. Conclusion: Inflammation-induced loss of CFTR-expressing ionocytes in airway cells from non-eosinophilic asthma may represent a key feature of disease pathogenesis and a novel drug target.

Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)—(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions.

Introduction Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. Methods pBECs from healthy participants (n = 5) were CR using γ-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult™ (PN-ALI) or Bronchial Epithelial Growth Medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza™) - (AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral load was assessed by RT-qPCR and anti-viral factors quantified by Legendplex following Rhinovirus-A1b (RVA1b) infection. Results CRpBECs differentiated in PneumaCult™ were smaller and had a lower TEER and cilia beat frequency (CBF) compared to BEGM media. PneumaCult™ media cultures exhibited significantly increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. Conclusion There are distinct structural and functional differences in CRpBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing and comparing CRpBECs ALI experiments.

Type 2 immune responses are characterized by elevated type 2 cytokines and blood eosinophilia. Emerging evidence suggests that people with chronic type 2 inflammatory lung diseases are not particularly susceptible to SARS-CoV-2 infection. Intriguingly, recent in vitro, ex vivo research demonstrates type 2 cytokines, particularly IL-13, reduce the risk of SARS-CoV-2 infection in the airway epithelium. IL-13 treatment in airway epithelial cells followed by SARS-CoV-2 diminished viral entry, replication, spread, and cell death. IL-13 reduces the expression of the angiotensin-converting enzyme 2 (ACE2) receptor in the airway epithelium and transmembrane serine protease 2 (TMPRSS2), particularly in ciliated cells. It also alters the cellular composition toward a secretory-cell-rich phenotype reducing total ciliated cells and, thus, reducing viral tropism. IL-13 enhances Muc5ac mucin and glycocalyx secretion in the periciliary layer, which acts as a physical barrier to restrict virus attachment. Moreover, type 2 airway immune cells, such as M2 alveolar macrophages, CD4+ tissue-resident memory T cells, and innate lymphoid 2 cells, may also rescue type 2 airways from SARS-CoV-2-induced adverse effects. In this review, we discuss recent findings that demonstrate how type 2 immunity alters immune responses against SARS-CoV-2 and its consequences on COVID-19 pathogenesis.

Introduction: Chronic obstructive pulmonary disease (COPD) is a complex and heterogeneous disease and is among the top three diseases in the world with respect to morbidity and mortality. Despite some advancements in treatment and understanding of the disease process in COPD, knowledge remains limited. This directed our attention towards exploring the gene expression profile of COPD patients to identify novel target genes that can help develop new therapies. Methods: Bronchial biopsies of non-smoking healthy (n=18), ex- and current-smoking COPD (n=56) patients with varying degrees of severity were collected, and bulk RNA sequencing was performed. Differential gene expression analysis was assessed using the edgeR package. We performed cellular-type deconvolution analysis on the different cell types of the airway epithelium using CIBERSORT method and removed any influence of cellular heterogeneity. Moreover, master regulators (MRs) driving COPD pathogenesis were identified using ARACNE and Viper packages in R. Results: We have performed genome-wide transcriptomic analysis of COPD patients relative to healthy individuals. We have initially identified 167 differentially expressed genes, of which 34, including AKR1B10,IRX4,CA12, LCN10 and TMIGD3 (logFC>1,FDR<0.05), were identified post-cellular composition correction, increasing confidence that they are associated with COPD. In addition, we identified several MRs, including ESRRA, TIGD5 and MXD3 (Differential activity>6.5, FDR< 0.01), involved in COPD progression. Conclusion: Our study reveals several novel candidate genes that might be involved in COPD development.

Although asthma and chronic obstructive pulmonary disease (COPD) are two distinct chronic airway inflammatory diseases, they often co-exist in a patient and the condition is referred to as asthma-COPD overlap (ACO). Lack of evidence regarding the inflammatory cells in ACO airways has led to their poor prognosis and treatment. The objective of this endobronchial biopsy (EBB) study was to enumerate inflammatory cellular changes in the airway wall of ACO compared with asthma, COPD current smokers (CS) and ex-smokers (ES), normal lung function smokers (NLFS), and non-smoker controls (HC). EBB tissues from 74 patients were immunohistochemically stained for macrophages, mast cells, eosinophils, neutrophils, CD8+ T-cells and CD4+ T-cells. The microscopic images of stained tissues were evaluated in the epithelium, reticular basement membrane (RBM) cells/mm RBM length, and lamina propria (LP) cells/mm2 up to a depth of 120 µM using the image analysis software Image-Pro Plus 7.0. The observer was blinded to the images and disease diagnosis. Statistical analysis was performed using GraphPad Prism v9. The tissue macrophages in ACO were substantially higher in the epithelium and RBM than in HC (P < 0.001 for both), COPD-ES (P < 0.001 for both), and -CS (P < 0.05 and < 0.0001, respectively). The ACO LP macrophages were significantly higher in number than COPD-CS (P < 0.05). The mast cell numbers in ACO were lower than in NLFS (P < 0.05) in the epithelium, lower than COPD (P < 0.05) and NLFS (P < 0.001) in RBM; and lower than HC (P < 0.05) in LP. We noted lower eosinophils in ACO LP than HC (P < 0.05) and the lowest neutrophils in both ACO and asthma. Furthermore, CD8+ T-cell numbers increased in the ACO RBM than HC (P < 0.05), COPD-ES (P < 0.05), and NLFS (P < 0.01); however, they were similar in number in epithelium and LP across groups. CD4+ T-cells remained lower in number across all regions and groups. These results suggest that the ACO airway tissue inflammatory cellular profile differed from the contributing diseases of asthma and COPD with a predominance of macrophages.

Rationale Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation.